Aspergillus ochraceus 11 alpha hydroxylase and oxidoreductase

ABSTRACT

The present invention relates to a novel cytochrome P450-like enzyme ( Aspergillus ochraceus  11 alpha hydroxylase) and an oxidoreductase ( Aspergillus ochraceus  oxidoreductase) isolated from cDNA library generated from the mRNA of  Aspergillus ochraceus  spores. When the cDNA encoding the 11 alpha hydroxylase was co-expressed in  Spodoptera frugiperda  (Sf-9) insect cells with the cDNA encoding human oxidoreductase as an electron donor, it successfully catalyzed the conversion of the steroid substrate 4-androstene-3,17-dione (AD) to 11 alpha-hydroxy-AD as determined by HPLC analysis. The invention also relates to nucleic acid molecules associated with or derived from these cDNAs including complements, homologues and fragments thereof, and methods of using these nucleic acid molecules, to generate, for example, polypeptides and fragments thereof. The invention also relates to the generation of antibodies that recognizes the  A. ochraceus  11 alpha hydroxylase and oxidoreductase and methods of using these antibodies to detect the presence of these native and recombinant polypeptides within unmodified and transformed host cells, respectively. The invention also provides methods of expressing the Aspergillus 11 alpha hydroxylase gene separately, or in combination with human or Aspergillus oxidoreductase, in heterologous host cells, to facilitate the bioconversion of steroid substrates to their 11 alpha hydroxy-counterparts.

PRIORITY

[0001] The present application claims priority under Title 35, UnitedStates Code, §119 of U.S. Provisional Application Serial No. 60/244,300,filed Oct. 30, 2000.

FIELD OF THE INVENTION

[0002] The present invention relates to a novel cytochrome P450-likeenzyme (Aspergillus ochraceus 11 alpha hydroxylase) and anoxidoreductase (Aspergillus ochraceus oxidoreductase) isolated from cDNAlibrary generated from the mRNA of Aspergillus ochraceus spores. Whenthe cDNA encoding the 11 alpha hydroxylase was co-expressed inSpodoptera frugiperda (Sf-9) insect cells with the cDNA encoding humanoxidoreductase as an electron donor, it successfully catalyzed theconversion of the steroid substrate 4-androstene-3,17-dione (AD) to 11alpha-hydroxy-AD as determined by HPLC analysis. The invention alsorelates to nucleic acid molecules associated with or derived from thesecDNAs including complements, homologues and fragments thereof, andmethods of using these nucleic acid molecules, to generate, for example,polypeptides and fragments thereof. The invention also relates to thegeneration of antibodies that recognize the A. ochraceus 11 alphahydroxylase and oxidoreductase and methods of using these antibodies todetect the presence of these native and recombinant polypeptides withinunmodified and transformed host cells, respectively. The invention alsoprovides methods of expressing the Aspergillus 11 alpha hydroxylase geneseparately, or in combination with human or Aspergillus oxidoreductase,in heterologous host cells, to facilitate the bioconversion of steroidsubstrates to their 11 alpha hydroxy-counterparts.

BACKGROUND OF THE INVENTION

[0003] Microbial transformation or bioconversion reactions have longbeen used to facilitate the chemical synthesis of a wide variety ofpharmaceutical products. Stereospecific reactions carried out under mildenzymatic conditions frequently offer advantages over comparablechemical processes which result in undesireable side products.Microorganisms also have the ability to carry out simultaneousindependent or sequential reactions on a substrate molecule, minimizingthe number of distinct steps in a synthesis and reducing the total costof the desired intermediate or end product.

[0004] General features of microbial systems used as biocatalysts forthe transformation of organic compounds has been reviewed (See e.g.,Goodhue, Charles T., Microb. Transform. Bioact. Compd., 1: 9-44, 1982).Biotransformations can be carried out, for example, in continuouscultures or in batch cultures. Enzymes secreted from the microorganismreact with a substrate, and the product can be recovered from themedium. Intracellular enzymes can also react with a substrate if it isable to enter cells by an active or a passive diffusion process.Immobilized, dried, permeabilized, and resting cells, and spores havealso been used for microbial transformations. The use of cell extractsand purified enzymes in solution, or immobilized on carriers, mayeventually offer significant cost or control advantages over traditionalfermentation methods.

[0005] Bioconversion reactions have been widely used in the field ofsteroids (Kieslich, K.; Sebek, O. K. Annu. Rep. Ferment. Processes 3:275-304, 1979; Kieslich, Klaus. Econ. Microbiol., 5 (Microb. EnzymesBioconvers.): 369-465, 1980). A variety of reactions have beencharacterized, including hydroxylation, epoxidation, oxidation,dehydrogenation, ring and side chain degradation, reduction, hydrolysis,and isomerization reactions. Many types of microorganisms have also beenused including species as diverse, for example, as Acremonium,Aspergillus, Rhizopus, Fusarium, Penicillium, Streptomyces, Actinomyces,Nocardia, Pseudomonas, Mycobacterium, Arthrobacter and Bacillus.

[0006] A variety of approaches have been used to facilitate thehydroxylation of intermediates used in the synthesis ofcommercially-important steroid compounds. U.S. Pat. No. 4,588,683, forexample, describes a method of preparing 11 beta, 17 alpha, 20, 21tetrahydroxy steroids by incubating substrate compounds in a mediumcomprising a fungal culture of the genus Curvularia capable of effecting11 beta hydroxylation. Aspergillus ochraceus cultures and preparationsof mycelia have also been used to convert progesterone and othersteroids to their corresponding 11 alpha hydroxy forms (Tan, L. andFalardeau, P., 1970; Tan L., and Falardeau P., J. Steroid Biochem. 1:221-227, 1970; Samanta, T. B. et al., Biochem. J. 176, 593-594, 1978;Jayanthi, C. R. et al., Biochem. Biophys. Res. Commun. 106: 1262-1268,1982).

[0007] The advent of new and expanded clinical uses of steroids for thetreatment of a wide variety of disorders has created a need for improvedmethods for the production of steroid compounds and their intermediateson a commercial scale. U.S. Pat. No. 4,559,332, for example, describes anumber of methods for the preparation of 20-spiroxane series of steroidcompounds, including methods for the preparation of eplerenone methylhydrogen 9,11α-epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate,γ-lactone (also referred to as eplerenone or epoxymexrenone) and relatedcompounds. WO 98/25948 and U.S. application Ser. No. 09/319,673 describenovel processes for the preparation of 9,11-epoxy steroid compounds,especially those of the 20-spiroxane series and their analogs, novelintermediates useful in the preparation of steroid compounds, andprocesses for the preparation of such novel intermediates. U.S. Pat. No.6,046,023 discloses improved methods for the microbial transformation ofcanrenone or estr-4-ene-3,17-dione into its 11 α-hydroxy analogue usingmicroorganisms of the genus Aspergillus, Rhizopus, and Pestelotia, usingsteroid substrates having a purity of less than 97% and more than 90% ata concentration greater than 10 g/L.

[0008] Many modern, systematic approaches needed to optimizebioconversion of particular steroid intermediates are often hindered byinsufficient biochemical knowledge of the enzymes involved in theirsynthesis and degradation. Eukaryotic cytochromes P450 appear to beassociated with the endoplasmic reticulum (ER) or mitochondrialmembranes. The electron donor for ER-associated cytochrome P450 enzymesis often an FAD/FMN-dependent NADPH-cytochrome P450 oxidoreductase.Electron transfer in the mitochondrial cytochromes P450 is usuallymediated by an NADPH-ferredoxin oxidoreductase and ferrodoxin. Thespecific electron donors known to be involved in mammaliansteroidogenesis, are also called adrenodoxin reductase and adrenodoxin,respectively.

[0009] While fungal biotransformations are known to be mediated bycytochrome P450 enzymes, many of these enzymes are extremely difficultto purify in an enzymatically-active form (van den Brink et al., FungalGenetics and Biology 23, 1-17, 1998). Many fungal P450 enzymes appear tobe associated with the endoplasmic reticulum (van den Brink et al.,Fungal Genetics and Biology 23, 1-17, 1998). Yeast have an adrenodoxinreductase homologue which was shown to couple with a mammalian 11 betahydroxylase in vitro. (Lacour et al., Journal of Biological Chemistry273, 23984-23992, 1998). In contrast, the electron donor which coupleswith Aspergillus ochraceus 11 alpha hydroxylase was predicted to be anNADPH-cytochrome P450 oxidoreductase (Samanta and Ghosh, J SteroidBiochem 28, 327-32, 1987). The steroid 11 alpha hydroxylation complex inRhizopus nigricans also appears to require an NADPH-cytochrome p450oxidoreductase (Makovec and Breskvar, Arch Biochem Biophys. 357, 310-6,1998). Amplification of cytochrome R. nigricans P450 andNADPH-cytochrome P450 reductase activities in preparations ofprogesterone-induced fungal mycelia may the facilitate biochemicalcharacterization of both enzymes (Makovec and Breskvar, PflugersArch—Eur J. Physiol 439(Suppl): R111-R112, 2000).

[0010]Aspergillus ochraceus spores have been shown to catalyze the 11alpha hydroxylation of steroid substrates such as progesterone (Dutta TK, Datta J, Samanta T B, Biochem. Biophys. Res. Commun. 192:119-123,1993). A. fumigatus is also known to exhibit a steroid 11 alphahydroxylase activity (Smith et al., J Steroid Biochem Mol Biol 49:93-100, 1994). The A. fumigatus enzyme is distinguished from the A.ochraceus enzyme, in that it appears to be a cytochrome P450 with dualsite-specificity for 11 alpha and 15 beta hydroxylation and, unlike theA. ochraceus hydroxylase, appears to be non-inducible.

[0011] Despite recent advances in sequencing technologies, detailedknowledge about the structural relationships of fungal cytochrome P450sgleaned from nucleotide sequence data remains primitive. Breskvar etal., (Biochem. Biophys. Res. Commun 1991; 178, 1078-1083, 1991) havedescribed a genomic DNA sequence from Rhizopus nigricans for a putativeP-450 encoding an 11α-hydroxylase for progesterone. This sequence maynot be complete, however, since the predicted amino acid sequence lacksthe canonical heme-binding motif, FxxGxxxCxG, which is common to almostall known cytochrome P-450 enzymes. (Nelson et al, Pharmacogenetics 6:1-42, 1996).

[0012] The cloning and characterization of the NADPH cytochrome P450oxidoreductase (cprA) gene of Aspergillus niger has been described (vanden Brink, J., et al., Genbank accession numbers Z26938, CAA81550, 1993,unpublished). The primary structure of Saccharomyces cerevisiaeNADPH-cytochrome P450 reductase has also been deduced from thenucleotide sequence of its cloned gene (Yabusaki et al., J. Biochem.103(6): 1004-1010, 1988).

[0013] Several other approaches have been used to facilitate the cloningand analysis of steroid enzymes. U.S. Pat. Nos. 5,422,262, 5,679,521,and European patent EP 0 528 906 B1, for example, describes theexpression cloning of steroid 5 alpha reductase, type 2. U.S. Pat. No.5,869,283, for example, describes an expression cassette comprisingheterologous DNAs encoding two or more enzymes, each catalyzing anoxidation step involved conversion of cholesterol into hydrocortisone,including the conversion of cholesterol to pregnenolone; the conversionof pregnenolone to progesterone; the conversion of progesterone to 17α-hydroxy-progesterone; the conversion of 17 α-hydroxyprogesterone tocortexolone; and the conversion of cortexolone to hydrocortisone.

[0014] The sequences of Aspergillus ochraceus 11 alpha hydroxylase andA. ochraceus oxidoreductase have not been reported. Knowledge abouttheir sequence could greatly facilitate the development of expressionvectors and recombinant host strains that can carry out more efficientbioconversions of steroid intermediates and the synthesis of endproducts on a commercial scale without the problems associated withpartially-characterized host strains or an incomplete understanding ofthe enzymes involved in steroidogenesis. The present invention overcomesmany of the limitations discussed above by identifying enzymes capableof carrying out the 11 alpha hydroxylation of steroids. This approachnot only greatly facilitates the use of 11 alpha hydroxylation, but alsopermits the development of new strategies for the identification ofsimilar enzymes from other fungi, the cloning of other enzymes involvedin steroidogenesis from Aspergillus ochraceus and other microorganisms,and the development of improved host strains or methods using free cellsor immobilized cells or enzymes in bioconversion reactions. Similarapproaches could also be developed to aid in the construction ofexpression vectors and recombinant host strains that are more amenableto propagation and control than wild-type microorganisms now commonlyused for bioconversion in large scale bioreactors.

SUMMARY OF THE INVENTION

[0015] In its broadest scope, the present invention provides a method toclone enzymes involved in steroid metabolism and use of these enzymes toproduce novel steroid intermediates and end-products. One aspect of theclaimed invention is to provide a novel enzyme 11 alpha hydroxylase andoxidoreductase, and their nucleic acids, proteins, peptides, fragments,and homologues. The invention also relates to methods of identifying andcloning other enzymes involved in steroid metabolism. The invention alsocovers novel vectors and host cells, a novel method for makingheterologous proteins by using the above vectors, and a method foridentifying the substrate specificity of the cloned enzymes.

[0016] The invention provides a means for determining the substratespecificity of the cloned 11 alpha hydroxylase, allelic variants,muteins, and fusion proteins thereof, permitting evaluation of a broadarray of steroid substrates including 3 keto delta 4,5 steroids (3 ketodelta 4 steroids); 3 keto delta 4,5 delta 6,7 steroids (3 keto delta 4delta 6 steroids); 3 keto delta 6,7 steroids (3 keto delta 6 steroids);or 3 keto delta 1,2 delta 4,5 steroids (3 keto delta 1 delta 4steroids). Preferred substrates for testing include (a) canrenone; (b)androstenedione; (c) aldona; (d) ADD (1,4 androstenedienedione) (e)mexrenone; (f) 6 beta mexrenone; (g) 9 alpha mexrenone; (h) 12 betamexrenone; (i) delta 12 mexrenone; (j) testosterone; (k) progesterone;(l) mexrenone 6,7-bis-lactone; and (m) mexrenone 7,9-bislactone.Preferably the cloned 11 alpha hydroxylase, allelic variants, muteins,and fusion proteins thereof do not also catalyze a second hydroxylationselected from the group consisting of 15 alpha or beta hydroxylation, 6alpha or beta hydroxylation, 7 alpha or beta hydroxylation, 9 alpha orbeta hydroxylation, 12 alpha or beta hydroxylation, and 17 alpha or betahydroxylation of substrates selected from the group consisting of 3 ketodelta 4,5 steroids; 3 keto delta 4,5 delta 6,7 steroids; or 3 keto delta6,7 steroids. Most preferably the cloned 11 alpha hydroxylase, allelicvariants, muteins, and fusion proteins thereof do not catalyze the 15beta hydroxylation of substrates selected from the group consisting of 3keto delta 4,5 steroids; 3 keto delta 4,5 delta 6,7 steroids; or 3 ketodelta 6,7 steroids.

[0017] The invention provides an isolated and purified nucleic acid,encoding Aspergillus ochraceus 11 alpha hydroxylase. It also provides anisolated DNA, cDNA, gene, and an allele of the gene encoding Aspergillusochraceus 11 alpha hydroxylase. Preferably the isolated and purifiednucleic acid is as set forth in SEQ ID NO: 01. Preferably the isolatedDNA, cDNA, gene, and an allele of the gene is as set forth in SEQ ID NO:01.

[0018] The invention provides an isolated protein having the amino acidsequence of Aspergillus ochraceus 11 alpha hydroxylase. It also providesan isolated variant of Aspergillus ochraceus 11 alpha hydroxylase, and afusion protein comprising this hydroxylase. Preferably the protein is asset forth in SEQ ID NO: 2. It also provides for variant of the proteinset forth in SEQ ID NO: 2.; a polypeptide which comprises SEQ ID NO: 2with at least one conservative amino acid substitution; polypeptides,with an amino acid sequence at least 99%, 95%, 90%, 75%, and 50%identical to SEQ ID NO: 2.

[0019] The invention provides an isolated and purified nucleic acid,encoding Aspergillus ochraceus 11 alpha oxidoreductase. It also providesan isolated DNA, cDNA, gene, and allele of the gene encoding Aspergillusochraceus oxidoreductase. Preferably, the isolated and purified nucleicacid, wherein said nucleic acid sequence is as set forth in SEQ ID NO:5. It also provides for an isolated DNA, cDNA, gene, and allele of thegene set forth in SEQ ID NO: 5.

[0020] The invention provides an isolated protein having the amino acidsequence of Aspergillus ochraceus oxidoreductase. It also provides anisolated variant of the protein having the amino acid sequence ofAspergillus ochraceus oxidoreductase, and a fusion protein comprisingthe amino acid sequence of Aspergillus ochraceus oxidoreductase.Preferably the isolated protein has the amino acid sequence set forth inSEQ ID NO: 6. It also provides an isolated variant of a protein setforth in SEQ ID NO: 6. a purified polypeptide, the amino acid sequenceof which comprises SEQ ID NO: 6 with at least one conservative aminoacid substitution; and a polypeptides with an amino acid sequence atleast 99%, 95%, 90%, 75%, and 50% identical to SEQ ID NO: 6.

[0021] The invention provides an isolated and purified nucleic acidencoding an enzyme that can catalyze the 11 alpha hydroxylation of 3keto delta 4,5 steroids (3 keto delta 4 steroids); 3 keto delta 4,5delta 6,7 steroids (3 keto delta 4 delta 6 steroids); 3 keto delta 6,7steroids (3 keto delta 6 steroids); or 3 keto delta 1,2 delta 4,5steroids (3 keto delta 1 delta 4 steroids). Preferably the enzyme doesnot catalyze the 15 beta hydroxylation of 3 keto delta 4,5 steroids; 3keto delta 4,5 delta 6,7 steroids; or 3 keto delta 6,7 steroids. Morepreferably, the hydroxylation is selected from the group consisting of:(a) canrenone to 11 alpha hydroxy canrenone; (b) androstenedione to 11alpha hydroxy androstenedione; (c) aldona to 11 alpha hydroxy aldona;(d) ADD (1,4 androstenedienedione) to 11 alpha hydroxy ADD; (e)mexrenone to 11 alpha hydroxy mexrenone; (f) 6 beta mexrenone to 11alpha hydroxy 6 beta mexrenone; (g) 9 alpha mexrenone to 11 alphahydroxy 9 alpha mexrenone; (h) 12 beta mexrenone to 11 alpha hydroxy 12beta mexrenone; (i) delta 12 mexrenone to 11 alpha hydroxy delta 12mexrenone; (j) testosterone to 11 alpha hydroxy testosterone; (k)progesterone to 11 alpha hydroxy progesterone; (l) mexrenone6,7-bis-lactone to 11 alpha hydroxy mexrenone 6,7-bis-lactone; and (m)mexrenone 7,9-bislactone to 11 alpha hydroxy mexrenone 7,9-bislactone.More preferably, the hydroxylation is selected from the group consistingof: (a) canrenone to 11 alpha hydroxy canrenone; (b) androstenedione to11 alpha hydroxy androstenedione; (c) aldona to 11 alpha hydroxy aldona;and (d) ADD (1,4 androstenedienedione) to 11 alpha hydroxy ADD. Mostpreferably the hydroxylation is from canrenone to 11 alpha hydroxycanrenone.

[0022] The invention also provides a method of expressing a protein thatcan catalyze the 11 alpha hydroxylation of 3 keto delta 4,5 steroids; 3keto delta 4,5 delta 6,7 steroids; 3 keto delta 6,7 steroids; or 3 ketodelta 1,2 delta 4,5 steroids comprising; (a) transforming ortransfecting host cells with an expression cassette comprising apromoter operably linked to a nucleic acid that encodes said protein,and (b) expressing said protein in said host cells. The invention alsoprovides for a method of producing the protein further comprising thestep of recovering said protein. Preferably, this protein is Aspergillusochraceus 11 alpha hydroxylase. More preferably, this method furthercomprises expressing an electron donor protein, wherein said electrondonor protein can donate electrons to said protein that can catalyze the11 alpha hydroxylation of 3 keto delta 4,5 steroids; 3 keto delta 4,5delta 6,7 steroids; 3 keto delta 6,7 steroids; or 3 keto delta 1,2 delta4,5 steroids. Preferably, the electron donor protein is selected fromthe group consisting of human oxidoreductase and Aspergillus ochraceusoxidoreductase. More preferably the electron donor protein isAspergillus ochraceus oxidoreductase. More preferably, the nucleic acidencoding said steroid 11 alpha hydroxylase and said electron donorprotein are on separate expression cassettes. More preferably, thenucleic acid encoding said steroid 11 alpha hydroxylase and saidelectron donor protein are on the same expression cassettes. Even morepreferably, the steroid 11 alpha hydroxylase is Aspergillus ochraceus 11alpha hydroxylase and said electron donor protein is humanoxidoreductase. Even more preferably, the steroid 11 alpha hydroxylaseis Aspergillus ochraceus 11 alpha hydroxylase and said electron donorprotein is Aspergillus ochraceus oxidoreductase. Preferably, theexpression cassette is on an expression vector. More preferably, theexpression vector is a baculovirus. Even more preferably, thebaculovirus is a nuclear polyhedrosis virus is selected from the groupconsisting of Autographa californica nuclear polyhedrosis virus andBombyx mori nuclear polyhedrosis virus. Most preferably, the nuclearpolyhedrosis virus is Autographa californica nuclear polyhedrosis virus.Preferably, the host cells are insect cells. More preferably, the insectcells are selected from the group consisting of Spodoptera frugiperda,Trichoplusia ni, Autographa californica, and Manduca sexta cells. Mostpreferably the insect cells are Spodoptera frugiperda cells. Theinvention also provides a for a method of expressing a protein whereinthe Aspergillus ochraceus 11 alpha hydroxylase is SEQ ID NO: 2; thehuman oxidoreductase is SEQ ID NO: 4; and the Aspergillus ochraceusoxidoreductase is SEQ ID NO: 6.

[0023] The invention also provides for an isolated and purifiedpolypeptide that can catalyze the 11 alpha hydroxylation of 3 keto delta4,5 steroids (3 keto delta 4 steroids); 3 keto delta 4,5 delta 6,7steroids (3 keto delta 4 delta 6 steroids); 3 keto delta 6,7 steroids (3keto delta 6 steroids); or 3 keto delta 1,2 delta 4,5 steroids (3 ketodelta 1 delta 4 steroids). Preferably, the polypeptide does not catalyzethe 15 beta hydroxylation of 3 keto delta 4,5 steroids; 3 keto delta 4,5delta 6,7 steroids; or 3 keto delta 6,7 steroids. More preferably, thehydroxylation is selected from the group consisting of: (a) canrenone to11 alpha hydroxy canrenone; (b) androstenedione to 11 alpha hydroxyandrostenedione; (c) aldona to 11 alpha hydroxy aldona; (d) ADD (1,4androstenedienedione) to 11 alpha hydroxy ADD; (e) mexrenone to 11 alphahydroxy mexrenone; (f) 6 beta mexrenone to 11 alpha hydroxy 6 betamexrenone; (g) 9 alpha mexrenone to 11 alpha hydroxy 9 alpha mexrenone;(h) 12 beta mexrenone to 11 alpha hydroxy 12 beta mexrenone; (i) delta12 mexrenone to 11 alpha hydroxy delta 12 mexrenone; (j) testosterone to11 alpha hydroxy testosterone; (k) progesterone to 11 alpha hydroxyprogesterone; (l) mexrenone 6,7-bis-lactone to 11 alpha hydroxymexrenone 6,7-bis-lactone; and (m) mexrenone 7,9-bislactone to 11 alphahydroxy mexrenone 7,9-bislactone. More preferably, the hydroxylation isselected from the group consisting of: (a) canrenone to 11 alpha hydroxycanrenone; (b) androstenedione to 11 alpha hydroxy androstenedione; (c)aldona to 11 alpha hydroxy aldona; and (d) ADD (1,4androstenedienedione) to 11 alpha hydroxy ADD. Most preferably thehydroxylation is from canrenone to 11 alpha hydroxy canrenone.

[0024] The invention also provides for an expression cassette comprisinga promoter operably linked to an isolated and purified nucleic acidencoding a polypeptide that can catalyze the 11 alpha hydroxylation of 3keto delta 4,5 steroids (3 keto delta 4 steroids); 3 keto delta 4,5delta 6,7 steroids (3 keto delta 4 delta 6 steroids); 3 keto delta 6,7steroids (3 keto delta 6 steroids); or 3 keto delta 1,2 delta 4,5steroids (3 keto delta 1 delta 4 steroids). More preferably, thehydroxylation is selected from the group consisting of: (a) canrenone to11 alpha hydroxy canrenone; (b) androstenedione to 11 alpha hydroxyandrostenedione; (c) aldona to 11 alpha hydroxy aldona; (d) ADD (1,4androstenedienedione) to 11 alpha hydroxy ADD; (e) mexrenone to 11 alphahydroxy mexrenone; (f) 6 beta mexrenone to 11 alpha hydroxy 6 betamexrenone; (g) 9 alpha mexrenone to 11 alpha hydroxy 9 alpha mexrenone;(h) 12 beta mexrenone to 11 alpha hydroxy 12 beta mexrenone; (i) delta12 mexrenone to 11 alpha hydroxy delta 12 mexrenone; (j) testosterone to11 alpha hydroxy testosterone; (k) progesterone to 11 alpha hydroxyprogesterone; (l) mexrenone 6,7-bis-lactone to 11 alpha hydroxymexrenone 6,7-bis-lactone; and (m) mexrenone 7,9-bislactone to 11 alphahydroxy mexrenone 7,9-bislactone. More preferably, the hydroxylation isselected from the group consisting of: (a) canrenone to 11 alpha hydroxycanrenone; (b) androstenedione to 11 alpha hydroxy androstenedione; (c)aldona to 11 alpha hydroxy aldona; and (d) ADD (1,4androstenedienedione) to 11 alpha hydroxy ADD. Most preferably thehydroxylation is from canrenone to 11 alpha hydroxy canrenone.

[0025] The invention also provides for an expression cassette comprisinga promoter operably linked to an isolated and purified nucleic acidencoding Aspergillus ochraceus oxidoreductase. Preferably the nucleicacid is SEQ ID NO: 6.

[0026] The invention also provides for an expression cassette comprisinga heterologous DNA encoding an enzyme from the metabolic pathway for thesynthesis of sitosterol to eplerenone wherein said enzyme catalyzes atleast one conversion selected from the group consisting of: (a)canrenone to 11 alpha hydroxy canrenone; (b) androstenedione to 11 alphahydroxy androstenedione; (c) aldona to 11 alpha hydroxy aldona; (d) ADD(1,4 androstenedienedione) to 11 alpha hydroxy ADD; (e) mexrenone to 11alpha hydroxy mexrenone; (f) 6 beta mexrenone to 11 alpha hydroxy 6 betamexrenone; (g) 9 alpha mexrenone to 11 alpha hydroxy 9 alpha mexrenone;(h) 12 beta mexrenone to 11 alpha hydroxy 12 beta mexrenone; (i) delta12 mexrenone to 11 alpha hydroxy delta 12 mexrenone; (j) testosterone to11 alpha hydroxy testosterone; and (k) progesterone to 11 alpha hydroxyprogesterone; (l) mexrenone 6,7-bis-lactone to 11 alpha hydroxymexrenone 6,7-bis-lactone; and (m) mexrenone 7,9-bislactone to 11 alphahydroxy mexrenone 7,9-bislactone and wherein the heterologous DNA isoperably linked to control sequences required to express the encodedenzymes in a recombinant host. Preferably the heterologous DNA codingsequences in the expression cassette are selected from the groupconsisting of the following genus and species: Aspergillus ochraceus,Aspergillus ochraceus, Aspergillus niger, Aspergillus nidulans, Rhizopusoryzae, Rhizopus stolonifer, Streptomyces fradiae, Bacillus megaterium,Pseudomonas cruciviae, Trichothecium roseum, Fusarium oxysporum Rhizopusarrhizus, Absidia coerula, Absidia glauca, Actinomucor elegans,Aspergillus flavipes, Aspergillus fumigatus, Beauveria bassiana,Botryosphaeria obtusa, Calonectria decora, Chaetomium cochliodes,Corynespora cassiicola, Cunninghamella blakesleeana, Cunninghamellaechinulata, Cunninghamella elegans, Curvularia clavata, Curvularialunata, Cylindrocarpon radicicola, Epicoccum humicola, Gongronellabutleri, Hypomyces chrysospermus, Monosporium olivaceum, Mortierellaisabellina, Mucor mucedo, Mucor griseocyanus, Myrothecium verrucaria,Nocardia corallina, Paecilomyces carneus, Penicillum patulum, Pithomycesatroolivaceus, Pithomyces cynodontis, Pycnosporium sp.,Saccharopolyspora erythrae, Sepedonium chrysospermum, Stachylidiumbicolor, Streptomyces hyqroscopicus, Streptomyces purpurascens,Syncephalastrum racemosum, Thamnostylum piriforme, Thielavia terricola,and Verticillium theobromae, Cephalosporium aphidicola, Cochlioboluslunatas, Tieghemella orchidis, Tieghemella hyalospora, Monosporiumolivaceum, Aspergillus ustus, Fusarium graminearum, Verticilliumglaucum, and Rhizopus nigricans. More preferably, the genus and speciesare selected from the group consisting of Aspergillus ochraceus,Aspergillus ochraceus, Aspergillus niger, Aspergillus nidulans, Rhizopusoryzae, Rhizopus stolonifer, Streptomyces fradiae, Bacillus megaterium,Pseudomonas cruciviae, Trichothecium roseum, Fusarium oxysporum,Rhizopus arrhizus, and Monosporium olivaceum. Most preferably, genus andspecies is Aspergillus ochraceus.

[0027] Preferably, the recombinant host cell and progeny thereofcomprise at least one expression cassette. More preferably, the host isa microorganism. Most preferably, the host is a bacterium. The inventionalso provides for a process for making one or more enzymes from themetabolic pathway for the transformation of sitosterol to eplerenonecomprising incubating the recombinant host cell in a nutrient mediumunder conditions where the one or more enzymes encoded by theheterologous DNA are expressed and accumulate. More preferably theprocess comprises the steps of: (a) incubating the compound to beoxidized in the presence the recombinant host cells under conditionswhere the compound is hydroxylated and the hydroxylated productaccumulates, and (b) recovering the hydroxylated product. Mostpreferably, the process comprises the steps of: (a) incubating thecompound to be oxidized in the presence of the enzymes produced underconditions where the compound is hydroxylated and the hydroxylatedproduct accumulates, and (b) recovering the hydroxylated product. Theinvention also provides for a host cells harboring an expressioncassette. More preferably the expression cassette is integrated into thechromosome of said host cell. More preferably, the expression cassetteis integrated into an expression vector.

[0028] The invention also provides for a method of determining thespecific activity of a cloned 11 alpha hydroxylase comprising the stepsof; (a) transforming host cells with an expression vector comprising anucleic acid that encodes said 11 alpha hydroxylase, (b) expressing said11 alpha hydroxylase in said host cells; (c) preparing subcellularmembrane fractions from said cells, (d) incubating said subcellularmembrane fractions with a steroid substrate, and (e) monitoringconversion of the steroid substrate to its 11 alpha hydroxy steroidcounterpart. Preferably, the further comprises transforming host cellswith an expression vector nucleic acid that encodes an oxidoreductase,and expressing said oxidoreductase in said host cells. More preferably,the oxidoreductase is human or Aspergillus ochraceus. Most preferablythe oxidoreductase is human oxidoreductase. Most preferably theoxidoreductase is Aspergillus ochraceus oxidoreductase.

[0029] The invention also provides for a protein having SEQ ID NO: 2 andvariants thereof that are at least 95% identical to SEQ ID NO: 2 andcatalyze the 11 alpha hydroxylation of 3 keto delta 4,5 steroids; 3 ketodelta 4,5 delta 6,7 steroids; 3 keto delta 6,7 steroids; or 3 keto delta1,2 delta 4,5 steroids, wherein said hydroxylation is selected from thegroup consisting of: (a) canrenone to 11 alpha hydroxy canrenone; (b)androstenedione to 11 alpha hydroxy androstenedione; (c) aldona to 11alpha hydroxy aldona; (d) ADD (1,4 androstenedienedione) to 11 alphahydroxy ADD; (e) mexrenone to 11 alpha hydroxy mexrenone; (f) 6 betamexrenone to 11 alpha hydroxy 6 beta mexrenone; (g) 9 alpha mexrenone to11 alpha hydroxy 9 alpha mexrenone; (h) 12 beta mexrenone to 11 alphahydroxy 12 beta mexrenone; (i) delta 12 mexrenone to 11 alpha hydroxydelta 12 mexrenone; (j) testosterone to 11 alpha hydroxy testosterone;and (k) progesterone to 11 alpha hydroxy progesterone. Preferably theenzyme does not catalyze the 15 beta hydroxylation of 3 keto delta 4,5steroids; 3 keto delta 4,5 delta 6,7 steroids; or 3 keto delta 6,7steroids.

[0030] The invention provides an isolated and purified nucleic acidencoding an enzyme that can catalyze the 11 alpha hydroxylation of 3keto delta 4,5 steroids (3 keto delta 4 steroids); 3 keto delta 4,5delta 6,7 steroids (3 keto delta 4 delta 6 steroids); 3 keto delta 6,7steroids (3 keto delta 6 steroids); or 3 keto delta 1,2 delta 4,5steroids (3 keto delta 1 delta 4 steroids) wherein the hydroxylation isselected from the group consisting of: (a) canrenone to 11 alpha hydroxycanrenone; (b) androstenedione to 11 alpha hydroxy androstenedione; (c)aldona to 11 alpha hydroxy aldona; (d) ADD (1,4 androstenedienedione) to11 alpha hydroxy ADD; (e) mexrenone to 11 alpha hydroxy mexrenone; (f) 6beta mexrenone to 11 alpha hydroxy 6 beta mexrenone; (g) 9 alphamexrenone to 11 alpha hydroxy 9 alpha mexrenone; (h) 12 beta mexrenoneto 11 alpha hydroxy 12 beta mexrenone; (i) delta 12 mexrenone to 11alpha hydroxy delta 12 mexrenone; (j) testosterone to 11 alpha hydroxytestosterone; and (k) progesterone to 11 alpha hydroxy progesterone.Preferably the enzyme does not catalyze the 15 beta hydroxylation of 3keto delta 4,5 steroids; 3 keto delta 4,5 delta 6,7 steroids; or 3 ketodelta 6,7 steroids.

[0031] The invention also provides for a purified polypeptide, the aminoacid sequence of which is selected from the group consisting of SEQ IDNO: 23, SEQ ID NO: 24, SEQ ID NO: 25.

[0032] The invention provides for a purified immunogenic polypeptide,the amino acid sequence of which comprises at least ten consecutiveresidues of SEQ ID NO: 2.

[0033] The invention provides for an isolated and purified antibodyhaving a binding specificity for 11 alpha hydroxylase having an aminoacid sequence as shown in SEQ ID NO: 2. Preferably the antibody binds toa protein region selected from the group consisting of (a) theN-terminal amino acids 1-10 of SEQ ID NO: 2; (b) the last 10 C-terminalamino acids of SEQ ID NO: 2; (c) amino acids SEQ ID NO: 23; (d) aminoacids SEQ ID NO: 24; and (e) amino acids SEQ ID NO: 25. Preferably theantibody is purified on a peptide column, wherein said peptide isselected from the group consisting of: (a) the N-terminal amino acids1-10 of SEQ ID NO: 2; (b) the last 10 C-terminal amino acids of SEQ IDNO: 2; (c) amino acids SEQ ID NO: 23; (d) amino acids SEQ ID NO: 24; and(e) amino acids SEQ ID NO: 25.

[0034] The invention also provides for a purified polypeptide, the aminoacid sequence of which is selected from the group consisting of SEQ IDNO: 26.

[0035] The invention also provides for a purified immunogenicpolypeptide, the amino acid sequence of which comprises at least tenconsecutive residues of SEQ ID NO: 6.

[0036] The invention also provides for an isolated and purified antibodyhaving a binding specificity for 11 alpha hydroxylase having an aminoacid sequence as shown in SEQ ID NO: 6. Preferably the antibody binds toa protein region selected from the group consisting of (a) theN-terminal amino acids 1-10 of SEQ ID NO: 6; (b) the last 10 C-terminalamino acids of SEQ ID NO: 6; and (c) amino acids SEQ ID NO: 26. Morepreferably, the antibody is purified on a peptide column, wherein saidpeptide is selected from the group consisting of: (a) the N-terminalamino acids 1-10 of SEQ ID NO: 6; (b) the last 10 C-terminal amino acidsof SEQ ID NO: 6; and (c) amino acids SEQ ID NO: 26.

[0037] The invention also provides for a composition comprising anantibody described above in an effective carrier, vehicle, or auxiliaryagent. It also provides for a composition comprising such an antibodyand a solution. The antibody may be a polyclonal antibody. The antibodymay also be a monoclonal antibody. The antibody may be conjugated to animmunoaffinity matrix. The invention also provides for a method of usingan immunoaffinity matrix to purify a polypeptide from a biological fluidor cell lysate. Preferably the immunoaffinity matrix is SEPHAROSE 4B.More preferably the method of using an immunoaffinity matrix to purify apolypeptide from a biological fluid or cell lysate uses SEPHAROSE 4B asan immunoaffinity matrix. More preferably, the method of using animmunoaffinity matrix to purify a polypeptide from a biological fluid orcell lysate uses SEPHAROSE 4B as an immunoaffinity matrix.

[0038] The invention also provides for a method of using a peptidecolumn to purify an antibody, wherein said peptide is selected from thegroup consisting of: (a) the N-terminal amino acids 1-10 of SEQ ID NO:2; (b) the last 10 C-terminal amino acids of SEQ ID NO: 2; (c) aminoacids SEQ ID NO: 23; (d) amino acids SEQ ID NO: 24; and (e) amino acidsSEQ ID NO: 25.

[0039] The invention also provides for a method of using a peptidecolumn to purify an antibody, wherein said peptide is selected from thegroup consisting of: (a) the N-terminal amino acids 1-10 of SEQ ID NO:6; (b) the last 10 C-terminal amino acids of SEQ ID NO: 6; and (c) aminoacids SEQ ID NO: 26.

[0040] The invention also provides for a method of detecting a firstpolypeptide in a biological fluid, wherein said first polypeptide isselected from the group consisting of 11 alpha hydroxylase andoxidoreductase, comprising the following steps: (a) contacting saidfluid with a second polypeptide, having a binding specificity for saidfirst polypeptide, and (b) assaying the presence of said secondpolypeptide to determine the level of said first polypeptide.Preferably, the second polypeptide is an antibody. More preferably, thesecond polypeptide is radiolabeled.

[0041] The invention also provides for a process for producing anisolated nucleic acid comprising hybridizing SEQ ID NO: 1 to genomic DNAin 6×SSC and 65° C. and isolating the nucleic acid detected with SEQ IDNO: 1. The invention also provides for an isolated DNA nucleic acidprepared according to this process.

[0042] The invention also provides for an isolated nucleic acid thatspecifically hybridizes under highly stringent conditions to thecomplement of the sequence set forth in SEQ ID NO: 1.

[0043] The invention also provides for a process for producing anisolated nucleic comprising hybridizing SEQ ID NO: 5 to genomic DNA in6×SSC and 65° C. and isolating the nucleic acid detected with SEQ ID NO:5. The invention also provides for an isolated DNA nucleic acid preparedaccording to this process.

[0044] The invention also provides for an isolated nucleic acid thatspecifically hybridizes under highly stringent conditions to thecomplement of the sequence set forth in SEQ ID NO: 5.

[0045] The invention also provides for a DNA construct which alters theexpression of a 11 alpha hydroxylase gene not normally expressed in acell when said DNA construct is inserted into chromosomal DNA of thecell, said DNA construct comprising: (a) a targeting sequence; (b) aregulatory sequence; and (c) the structural gene for a steroid 11 alphahydroxylase. The invention also provides for a host cell harboring thisDNA construct.

[0046] The invention also provides for a DNA construct which alters theexpression of a 11 alpha hydroxylase gene not normally expressed in acell when said DNA construct is inserted into chromosomal DNA of thecell, said DNA construct comprising: (a) a targeting sequence; (b) aregulatory sequence; and (c) the structural gene for a steroidoxidoreductase. The invention also provides for a host cell harboringthis DNA construct.

[0047] The invention also provides for use of a host cell harboring acloned 11 alpha hydroxylase for the manufacture of a medicament fortherapeutic application to treat heart disease, inflammation, arthritis,or cancer.

[0048] The invention also provides for a composition comprising fromabout 0.5-to about 500 g/L molasses, 0.5-50 g/L cornsteep liquid, 0.5-50g/L KH₂PO₄, 2.5-250 g/L NaCl, 2.5-250 g/L glucose, and 0.04-4 g/Lprogesterone, pH 3.5-7. Preferably, this composition is comprised offrom about 10-250 g/L molasses, 1-25 g/L cornsteep liquid, 1-25 g/LKH₂PO₄, 5-125 g/L NaCl, 5-125 g/L glucose, and 0.08-2 g/L progesterone,pH 4.5-6.5. More preferably, the composition is comprised of from about25-100 g/L molasses, 2.5-10 g/L cornsteep liquid, 2.5-10 g/L KH₂PO₄,12.5-50 g/L NaCl, 12.5-50 g/L glucose, and 0.2-0.8 g/L progesterone, pH5.5-6.0. Most preferably the composition comprises about 50 g/Lmolasses, 5 g/L cornsteep liquid, 5 g/L KH₂PO₄, 25 g/L NaCl, 25 g/Lglucose, 20 g/L agar, and 0.4 g/L progesterone, pH 5.8.

[0049] The invention also provides for a semisolid formulation of any ofthe compositions described above, further comprising from about 4-100g/L agar. Preferably the agar is at a concentration of from about 10-40g/L agar. More preferably, the agar is about 20 g/L agar.

[0050] The invention also provides for the use of any of thecompositions describe above to produce spores from the microorganismselected from the group consisting of Aspergillus ochraceus, Aspergillusniger, Aspergillus nidulans, Rhizopus oryzae, Rhizopus stolonifer, andTrichothecium roseum, Fusarium oxysporum Rhizopus arrhizus, Monosporiumolivaceum. Penicillum chrysogenum, and Absidia coerula. Preferably, thecomposition is used to produce spores from Aspergillus ochraceus.

Definitions

[0051] The following is a list of abbreviations and the correspondingmeanings as used interchangeably herein:

[0052] 11 alpha hydroxycanrenone=11 alphahydroxy-4-androstene-3,17-dione (C₂₂H₂₈O₄, MW 356.46)

[0053] AcNPV=Autographa californica nuclear polyhedrosis virus, a memberof the Baculoviridae family of insect viruses

[0054] AD=androstenedione or 4-androstene-3,17-dione (C₂₂H₂₈O₃, MW340.46)

[0055] aldadiene=canrenone

[0056] Amp=ampicillin

[0057] attTn7=attachment site for Tn7 (a preferential site for Tn7insertion into bacterial chromosomes)

[0058] bacmid=recombinant baculovirus shuttle vector isolated from E.coli

[0059] Bluo-gal=halogenated indolyl-β-D-galactoside

[0060] bp=base pair(s)

[0061] Cam=chloramphenicol

[0062] cDNA=complementary DNA

[0063] DMF═N,N-dimethylformamide

[0064] ds=double-stranded

[0065] eplerenone or epoxymexrenone=methyl hydrogen9,11α-epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-lactone(MW 414.5)

[0066] g=gram(s)

[0067] Gen=gentamicin

[0068] hoxr=human oxidoreductase

[0069] HPLC=high performance liquid chromatography

[0070] hydroxycanrenone=11 alpha- or 11 beta-hydroxycanrenone

[0071] IPTG=isopropyl-β-D-thiogalactopyranoside

[0072] Kan=kanamycin

[0073] kb=kilobase(s), 1000 bp(s)

[0074] mb=megabase(s)

[0075] Me=methyl

[0076] mg=milligram(s)

[0077] ml or mL=milliliter(s)

[0078] mm=millimeter

[0079] mM=millimolar

[0080] NMR=nuclear magnetic resonance

[0081] oxr=oxidoreductase

[0082] PCR=polymerase chain reaction

[0083] r=resistant or resistance

[0084] RP-HPLC=reverse phase high performance liquid chromatography

[0085] RT=room temperature

[0086] RT-PCR=reverse transcriptase polymerase chain reaction

[0087] s=sensitive

[0088] SDS-PAGE=sodium dodecyl sulfate polyacrylamide gelelectrophoresis

[0089] Spc/Str=spectinomycin/streptomycin

[0090] Tet=tetracycline

[0091] Tn=transposon

[0092] ts=temperature-sensitive

[0093] U=units

[0094] ug or μg=microgram(s)

[0095] ul or μl=microliter(s)

[0096] X-gal 5-bromo-3-chloro-indolyl-β-D-galactopyranoside

[0097] X-gluc=5-bromo-3-chloro-indolyl-β-D-glucopyranoside

[0098] The following is a list definitions of various terms used herein:

[0099] The species “Aspergillus ochraceus NRRL 405” means thefilamentous fungus Aspergillus ochraceus NRRL 405, accession number18500, obtained from the American Type Culture Collection (ATCC). A.ochraceus NRRL 405 and A. ochraceus ATCC 18500 are the same strain,catalogued differently.

[0100] The term “amino acid(s)” means all naturally occurring L-aminoacids, including norleucine, norvaline, homocysteine, and ornithine.

[0101] The term “degenerate” means that two nucleic acid moleculesencode for the same amino acid sequences but comprise differentnucleotide sequences.

[0102] The term “fragment” means a nucleic acid molecule whose sequenceis shorter than the target or identified nucleic acid molecule andhaving the identical, the substantial complement, or the substantialhomologue of at least 10 contiguous nucleotides of the target oridentified nucleic acid molecule.

[0103] The term “fusion protein” means a protein or fragment thereofthat comprises one or more additional peptide regions not derived fromthat protein.

[0104] The term “probe” means an agent that is utilized to determine anattribute or feature (e.g. presence or absence, location, correlation,etc.) of a molecule, cell, tissue, or organism.

[0105] The term “promoter” is used in an expansive sense to refer to theregulatory sequence(s) that control mRNA production. Such sequencesinclude RNA polymerase binding sites, enhancers, etc.

[0106] The term “protein fragment” means a peptide or polypeptidemolecule whose amino acid sequence comprises a subset of the amino acidsequence of that protein.

[0107] The term “recombinant” means any agent (e.g., DNA, peptide,etc.), that is, or results from, however indirectly, human manipulationof a nucleic acid molecule.

[0108] The term “selectable or screenable marker genes” means geneswhose expression can be detected by a probe as a means of identifying orselecting for transformed cells.

[0109] The term “specifically bind” means that the binding of anantibody or peptide is not competitively inhibited by the presence ofnon-related molecules.

[0110] The term “specifically hybridizing” means that two nucleic acidmolecules are capable of forming an anti-parallel, double-strandednucleic acid structure.

[0111] The term “substantial complement” means that a nucleic acidsequence shares at least 80% sequence identity with the complement.

[0112] The term “substantial fragment” means a nucleic acid fragmentwhich comprises at least 100 nucleotides.

[0113] The term “substantial homologue” means that a nucleic acidmolecule shares at least 80% sequence identity with another.

[0114] The term “substantially hybridizing” means that two nucleic acidmolecules can form an anti-parallel, double-stranded nucleic acidstructure under conditions (e.g., salt and temperature) that permithybridization of sequences that exhibit 90% sequence identity or greaterwith each other and exhibit this identity for at least about acontiguous 50 nucleotides of the nucleic acid molecules.

[0115] The term “substantially-purified” means that one or moremolecules that are or may be present in a naturally-occurringpreparation containing the target molecule will have been removed orreduced in concentration.

[0116] The following is a list of steroids, corresponding terms, andtheir structures, as used interchangeably herein: CA Index # Name Name:Other Names Formula Structure 1 Eplerenone Pren-4-ene-7,21-di-carboxylic acid, 9,11-epoxy-17-hy- droxy-3-oxo, γ-lactone, methyl ester,(7α,11α,17α)-(9CI) Spiro[9,11-e- poxy-9H-cyclo- penta[a]phenan-threne-17(2H), 2′(3′H)-furan], pregn-4-ene-7,21-di- carboxylic acidderv.; CGP 30083; Eplerenone; #SC 66110 C24H30O6

2 Aldadiene; Canrenone Pregna-4,6-diene-21-car- boxy- lic acid,17-hydroxy-3-oxo, γ-lactone, (17α)-(9CI) 17α-Pregna-4,6-diene-21-car-boxylic acid, 17-hydroxy-3-oxo-, γ-lactone(6CI, 7CI, 8CI);Spiro[17H-cyclo- penta[a]phenanthrene-17,2′(5′H)-furan],pregna-4,6-diene-21-car- # boxylic acid deriv.; 11614 R.P.;17β-Hydroxy-3-oxo- pregna-4,6-diene-21-carboxylic acid; 20-Spi-roxa-4,6-diene-3,21-dione; Aldadiene; Canrenone; Phanurane; SC 9376;Spirolactone SC 14266 C22H28O3

3 11α-Hydroxy- canrenone Pregna-4,6-diene-21-carboxy- lic acid,11,17-di- hydroxy-3-oxo, γ-lac- tone, (11α,17α)-(9CI)11α-Hydroxycanrenone C22H28O4

5 Aldone ethyl enol ether Pregna-4,6-diene-21-carboxy- lic acid,3-ethoxy-17-hy- droxy-, γ-lactone(9CI) Spiro[17H-cyclo-penta[a]phenanthrene-17,2′(5′H)-furan], pregna-4,6-diene-21-carboxy- licacid deriv.; Aldona ethyl enol ether C24H34O3

6 Androstenedione Androst-4-ene-3,17-dione (8CI, 9CI)Δ4-Androstene-3,17-dione; 17-Ketotestosterone; 3,17-Dioxoandrost-4-ene;Androstenedione; Fecundin; SKF 2170 C19H26O2

7 11α-Hydroxy- androstenedione Androst-4-ene-3,17-dione, 11-hydroxy-,(11α)-(9CI) Androst-4-ene-3,17-dione, 11α-hydroxy- (8CI); 11α-Hydroxy-androstendione; 11α-Hydroxy- androstenedione C19H26O3

8 Mexrenone Pregn-4-ene-7,21-di- carboxylic acid, 17-hydroxy-3-oxo,γ-lactone, methyl ester, (7α,17α)-(9CI) Spiro[17H-cyclo-penta[a]phenanthrene-17,2′(5′H)-furan], pregn-4-ene-7,21-dicarboxylicacid deriv.; Mexrenone; SC 25152; ZK 32055 C24H32O5

9 11β-Hydroxy- mexrenone Pregn-4-ene-7,21-di- carboxylic acid,11,17-dihydroxy-3-oxo, γ-lactone, methyl ester, (7α11β,17α)-(9CI)11β-Hydroxy- mexrenone C24H32O6

10 12β-Hydroxy- mexrenone Pregn-4-ene-7,21-di- carboxylic acid,12,17-dihydroxy-3-oxo, γ-lactone, methyl ester, (7α,12β,17α)-(9CI)12β-Hydroxy- mexrenone C24H32O6

11 9α-Hydroxy- mexrenone Pregn-4-ene-7,21-di- carboxylic acid,9,17-dihydroxy-3-oxo-, 21,17-lactone, 7-methyl ester, (7α,17α)-(9CI)9α-Hydroxy- mextrenone C24H32O6

12 6β-Hydroxy- mexrenone Pregn-4-ene-7,21-di- carboxylic acid,6,17-dihydroxy-3-oxo, γ-lactone, methyl ester, (6β,7α,17α)-(9CI)Spiro[17H-cyclo- penta[a]phenan- threne-17,2′(3′H)-furan],pregn-4-ene-7,21-di- carboxylic acid deriv.; 6β-HydroxymextrenoneC24H32O6

13 Progesterone Pregn-4-ene-3,20-dione (9CI) Progesterone (8CI);Δ4-Pregnene-3,20-dione; and >70 other names C21H30O2

14 Ester-4-ene-3,17-dione Estr-4-ene-3,17-dione (6CI, 8CI, 9CI)(+)-19-Norandrost-4-ene-3,17-dione; Δ4-Estrene-3,17-dione;19-Norandrost-4-ene-3,17-dione C18H24O2

15 delta 1,4-androstadiene-3,17-dione (ADD)Androsta-1,4-diene-3,17-dione (7CI, 8CI, 9CI)Δ1,4-Androstadiene-3,17-dione; 1-Dehydroandrostenedione;Androstadienedione; Androstane-1,4-diene-3,17-dione C19H24O2

16 11α-Hydroxy- androsta-1,4-diene-3,17-dione (11 alpha hydroxy ADD)Androsta-1,4-diene-3,17-dione, 11-hydroxy-, (11α)-(9CI)Androsta-1,4-diene-3,17-dione, 11α-hydroxy- (6CI, 7CI, 8CI);11α-Hydroxy- androsta-1,4-diene-3,17-dione; Kurchinin C19H24O3

17 aldona Compound 5 (aldona ethyl enol ether) with O═ in place of EtO—at position 3 18 mextrenone Compound 12 with cyclic bis-lactone6,7-bislactone ring(—O—C═O—) formed between carbons at positions 6 and 7(See U.S. Pat. No. 5,981,744 for discussion of similar lactone rings) 1911 alpha 11 alpha hydroxy version of hydroxy Compound 18 mexrenone6,7-bislactone 20 mexrenone Compound 11 with cyclic bis-lactone7,9-bislactone ring (—O—C═O—) formed between carbons at positions 7 and9 (See U.S. Pat. No. 5,981,744 for discussion of similar lactone rings)21 11 alpha 11 alpha hydroxy version of hydroxy Compound 20 mexrenone7,9-bislactone

[0117]FIG. 1—Nucleotide and Protein Sequence of Aspergillus ochraceus 11Alpha Hydroxylase

[0118] The nucleotide and protein sequences of Aspergillus ochraceus 11alpha hydroxylase (SEQ ID NO: 1, SEQ ID NO: 2, respectively) aredisplayed.

[0119]FIG. 2—Nucleotide and Protein Sequence of Human Oxidoreductase

[0120] The nucleotide and protein sequences of human oxidoreductase (SEQID NO: 3, SEQ ID NO: 4, respectively) are displayed. The predicted aminoacid sequence of human oxidoreductase independently cloned from a cDNAlibrary prepared by RT-PCR using the RNA from a human HepG2 cells as atemplate, as disclosed in this specification, matches that previousreported by three different laboratories. The GenBank accession numbersfor these loci include A60557 (NADPH—ferrihemoprotein reductase (EC1.6.2.4)-human); AAG09798 (NADPH-cytochrome P450 reductase [Homosapiens]), and P16435 (NADPH-CYTOCHROME P450 REDUCTASE (CPR) (P450R)).

[0121] The amino acid sequence of AAB21814 (cytochrome P450 reductase{EC 1.6.2.4} [human, placenta, Peptide Partial, 676 aa]), differs fromhuman oxidoreductase A60557 and P16435 at 4 residues: A→V at 500, F→L at518, V→W at 537, and A→H at 538. The initial methionine is also missingfrom AAB21814. The cognate nucleic acid for AA21814 (S90469 [cytochromeP450 reductase [human, placenta, mRNA Partial, 2403 nt]) lacks the ATGcodon for the initial methionine and includes a C→T change at 1496, aC→A, change at 1551, and a frameshift due to a missing G at 1605 whichis resolved by the addition of a T at 1616.

[0122] References for these loci are as follows: A60557 [Yamano, S.,Aoyama, T., McBride, O. W., Hardwick, J. P., Gelboin, H. V. andGonzalez, F. J. Human NADPH—P450 oxidoreductase: complementary DNAcloning, sequence and vaccinia virus-mediated expression andlocalization of the CYPOR gene to chromosome 7 Mol. Pharmacol. 36 (1),83-88 (1989)]; AAG09798 [Czerwinski, M., Sahni, M., MadanA. andParkinson, A. Polymorphism of human CYPOR: Expression of new allele.Unpublished, Direct Submission], and P16435 [Haniu, M., McManus, M. E.,Birkett, D. J., Lee, T. D. and Shively, J. E. Structural and functionalanalysis of NADPH-cytochrome P-450 reductase from human liver: completesequence of human enzyme and NADPH-binding sites. Biochemistry 28 (21),8639-8645 (1989]]; AAB21814 [Shephard, E. A., Palmer, C. N., Segall, H.J. and Phillips, I. R. Quantification of cytochrome P450 reductase geneexpression in human tissues. Arch. Biochem. Biophys. 294 (1), 168-172(1992)]; S90469 [Shephard, E. A., Palmer, C. N., Segall, H. J. andPhillips, I. R. Quantification of cytochrome P450 reductase geneexpression in human tissues. Arch. Biochem. Biophys. 294 (1), 168-172(1992)].

[0123]FIG. 3—Nucleotide and Protein Sequence of Aspergillus ochraceusOxidoreductase

[0124] The nucleotide and protein sequences of Aspergillus ochraceus 11oxidoreductase (SEQ ID NO: 5, SEQ ID NO: 6, respectively) are displayed.

[0125]FIG. 4—Amino Acid Homology Alignment of A. ochraeeus 11 AlphaHydroxylase with the Top 10 BLAST Hits from GenBank

[0126]Aspergillus ochraceus steroid 11 alpha hydroxylase (SEQ ID NO:02), cloned into plasmid pMON45624 (SEQ ID NO: 01), was aligned withrelated enzymes found in GenBank using the BLASTP program thatimplements a heuristic matching algorithm (Altschul et al., J Mol BiolOct 5;215(3):403-10, 1990). The GenBank accession numbers (its probablefunction, [genus and species]) for the top 10 matches are as follows:CAA75565 (cytochrome P450 monooxygenase [Gibberella fujikuroi]; CAB91316(probable cytochrome P450 monooxygenase (lovA) [Neurospora crassa]);CAB56503 (cytochrome P450 [Catharanthus roseus]); AAB94588 (CYP71D10p[Glycine max]); CAA75566 (cytochrome P450 monooxygenase [Gibberellafujikuroi]); AAD34552 (cytochrome P450 monooxygenase [Aspergillusterreus]); CAA75567 (cytochrome P450 monooxygenase [Gibberellafujikuroi]); CAA76703 (cytochrome P450 [Gibberella fujikuroi]); CAA57874(unnamed protein product [Fusarium oxysporum]); CAA91268 (similar tocytochrome P450-cDNA EST yk423b11.3 comes from this gene [Caenorhabditiselegans ]).

[0127] References for these loci are as follows: CAA75565 [Tudzynski, B.and Holter, K., Gibberellin biosynthetic pathway in Gibberellafujikuroi: evidence for a gene cluster. Fungal Genet. Biol. 25 (3),157-170 (1998)]; CAB91316 [Schulte, U., Aign, V., Hoheisel, J., Brandt,P., Fartmann, B., Holland, R., Nyakatura, G., Mewes, H. W. andMannhaupt, G., Unpublished]; CAB56503 [Schroeder, G., Unterbusch, E.,Kaltenbach, M., Schmidt, J., Strack, D. and Schroeder, J. Light-inducedcytochrome P450-dependent enzyme in indole alkaloid biosynthesis:tabersonine 16-hydroxylase FEBS Lett. 458, 97-102 (1999)]; AAB94588[Siminszky, B., Corbin, F. T., Ward, E. R., Fleischmann, T. J. andDewey, R. E. Expression of a soybean cytochrome P450 monooxygenase cDNAin yeast and tobacco enhances the metabolism of phenylurea herbicides.Proc. Natl. Acad. Sci. U.S.A. 96 (4), 1750-1755 (1999)]; CAA75566[Tudzynski, B. and Holter, K. Gibberellin biosynthetic pathway inGibberella fujikuroi: evidence for a gene cluster. Fungal Genet. Biol.25 (3), 157-170 (1998)]; AAD34552 [Kennedy, J., Auclair, K., Kendrew, S.G., Park, C., Vederas, J. C. and Hutchinson, C. R. Accessory ProteinsModulate Polyketide Synthase Activity During Lovastatin Biosynthesis.Science (1999) In press]; CAA75567 [Tudzynski, B. and Holter, K.Gibberellin biosynthetic pathway in Gibberella fujikuroi: evidence for agene cluster. Fungal Genet. Biol. 25 (3), 157-170 (1998)]; CAA76703[Tudzynski, B. and Hoelter, K. Characterization of P450 monooxygenasegenes from Gibberella fujikuroi. Unpublished]; CAA57874 [Mouyna, I. andBrygoo, Y. Disruption of a Fusarium oxysporum f.sp. elaeidis cytochromeP450 gene by a repetitive sequence. Unpublished]; and CAA91268 [NoAuthors. Genome sequence of the nematode C. elegans: a platform forinvestigating biology. The C. elegans Sequencing Consortium. Science 282(5396), 2012-2018 (1998) [Published errata appear in Science Jan. 1,1999;283(5398):35 and Mar. 26, 1999;283(5410):2103 and Sep. 3,1999;285(5433):1493]]].

[0128]FIG. 5—Phylogenetic Tree Showing the Relatedness of Aspergillusochraceus 11 Alpha Hydroxylase to the Top 10 BLAST Hits from GenBank

[0129] A phylogenetic tree displaying the genetic relatedness ofAspergillus ochraceus steroid 11 alpha hydroxylase, cloned into plasmidpMON45624, was aligned with related enzymes found in GenBank. BLAST wasused to find the related enzymes within GenBank, and ClustalW was usedgenerate the multiple sequence alignment and phylogenetic tree depictedin this figure. Descriptions of the GenBank accession numbers used aslabels in the figure are the same as that described above for the legendto FIG. 4.

[0130]FIG. 6—Percent Homology Between Aspergillus ochraceus 11 AlphaHydroxylase and the Top 10 BLAST Hits from GenBank

[0131] The percent homology between Aspergillus ochraceus steroid 11alpha hydroxylase and the top 10 enzymes found in GenBank using BLASTwas calculated using CLUSTAL (Thompson et al., Comput. Appl. Biosci.10:19-29, 1994).

[0132]FIG. 7—Amino Acid Homology Alignment of Aspergillus ochraceus andHuman Oxidoreductase to NADPH Cytochrome P450 Reductases from A. niger,Mouse, and S. cerevisiae

[0133] The amino acid sequences of Aspergillus ochraceus steroidoxidoreductase (SEQ ID NO: 06) cloned into plasmid pMON45632 (SEQ ID NO:05), and human oxidoreductase (SEQ ID NO: 03), cloned into plasmidpMON45605 (SEQ ID NO: 04) were aligned with related enzymes from A.niger, mouse, and S. cervisiase, as described above. The GenBankaccession numbers (probable function, [genus and species]) are asfollows: BAA02936 (NADPH-cytochrome P450 reductase precursor[Saccharomyces cerevisiae ]); CAA81550 NADPH cytochrome P450oxidoreductase [Aspergillus niger ]; P16435 (NADPH-CYTOCHROME P450REDUCTASE (CPR) (P450R) [human]); BAA04496 (NADPH-cytochrome P450oxidoreductase [Mus musculus]).

[0134] References for these loci are as follows: BAA02936 [Yabusaki, Y.,Murakami, H. and Ohkawa, H. Primary structure of Saccharomycescerevisiae NADPH-cytochrome P450 reductase deduced from nucleotidesequence of its cloned gene. J. Biochem. 103 (6), 1004-1010 (1988)];CAA81550 [van den Brink, J., van Zeijl, C., van den Hondel, C. and vanGorcom, R. Cloning and characterization of the NADPH cytochrome P450oxidoreductase (cprA) gene of Aspergillus niger. Unpublished]; P16435[Haniu, M., McManus, M. E., Birkett, D. J., Lee, T. D. and Shively, J.E. Structural and functional analysis of NADPH-cytochrome P-450reductase from human liver: complete sequence of human enzyme andNADPH-binding sites Biochemistry 28 (21), 8639-8645 (1989)]; BAA04496[Ohgiya, S., Shinriki, N., Kamataki, T. and Ishizaki, K. MouseNADPH-cytochrome P-450 oxidoreductase: molecular cloning and functionalexpression in yeast. Biochim. Biophys. Acta 1186 (1-2), 137-141 (1994)].

[0135]FIG. 8—Amino Acid Homology Alignment of A. ochraceusOxidoreductase to NADPH Cytochrome P450 Reductases from A niger, Mouse,and S. cerevisiae

[0136] The amino acid sequence of Aspergillus ochraceus steroidoxidoreductase (SEQ ID NO: 06) cloned into plasmid pMON45632 (SEQ ID NO:05), was aligned with related fungal enzymes from A. niger and S.cervisiase, as described above. Descriptions of the GenBank accessionnumbers used as labels in the figure are the same as that describedabove for the legend to FIG. 7, above.

[0137]FIG. 9—Phylogenetic Tree Showing the Relatedness of Aspergillusochraceus and Human Oxidoreductase to Reductases from A. niger, Yeast,and Mouse.

[0138] A phylogenetic tree displaying the genetic relatedness ofAspergillus ochraceus oxidoreductase (SEQ ID NO: 06), cloned intoplasmid pMON45632 (SEQ ID NO: 05), was aligned with related enzymes.BLAST was used to find the related enzymes within GenBank, and ClustalWwas used generate the multiple sequence alignment and phylogenetic treedepicted in this figure. Descriptions of the GenBank accession numbersused as labels in the figure are the same as that described above forthe legend to FIG. 7, above.

[0139]FIG. 10—Percent Identity Between Aspergillus ochraceusOxidoreductase and Reductases from A. niger, Yeast, and Mouse.

[0140] The percent identity between Aspergillus ochraceus oxidoreductaseand the oxidoreductases from A. niger, yeast, and mouse was calculatedusing Clustal W and Boxshade.

[0141]FIG. 11—Alignment of Human Oxidoreductase with Top 4 Hits fromSwissProt

[0142] The amino acid sequences of human steroid oxidoreductase (SEQ IDNO: 04), cloned into plasmid pMON45605 (SEQ ID NO: 03), whichcorresponds to the amino acid sequence of the corrected sequencereported for P16435 below, was aligned with the top 4 hits from theSWISSPROT protein sequence database, as described above. The SWISSPROTaccession numbers {locus} [common name] and species]) probable function)are as follows: P16435 {NCPR_HUMAN} [human] NADPH-CYTOCHROME P450REDUCTASE; P00389 {NCPR_RABIT} [rabbit] NADPH-CYTOCHROME P450 REDUCTASE;P00388 {NCPR_RAT} [rat] NADPH-CYTOCHROME P450 REDUCTASE; P37040{NCPR_MOUSE} [mouse] NADPH-CYTOCHROME P450 REDUCTASE; PO₄₁₇₅ {NCPR_PIG}[pig] (NADPH-CYTOCHROME P450 REDUCTASE.

[0143] References for these loci are as follows: P16435 [Haniu, M.,McManus, M. E., Birkett, D. J., Lee, T. D. and Shively, J. E. Structuraland functional analysis of NADPH-cytochrome P-450 reductase from humanliver: complete sequence of human enzyme and NADPH-binding sites.Biochemistry 28 (21), 8639-8645 (1989)]; P00389 [Katagiri, M., Murakami,H., Yabusaki, Y., Sugiyama, T., Okamoto, M., Yamano, T. and Ohkawa, H.Molecular cloning and sequence analysis of full-length cDNA for rabbitliver NADPH-cytochrome P-450 reductase mRNA. J. Biochem. 100 (4),945-954 (1986)]; P00388 [Porter, T. D. and Kasper, C. B. Codingnucleotide sequence of rat NADPH-cytochrome P-450 oxidoreductase cDNAand identification of flavin-binding domains. Proc. Natl. Acad. Sci.U.S.A. 82 (4), 973-977 (1985)]; P37040 [Ohgiya, S., Shinriki, N.,Kamataki, T. and Ishizaki, K. Mouse NADPH-cytochrome P-450oxidoreductase: molecular cloning and functional expression in yeast.Biochim. Biophys. Acta 1186 (1-2), 137-141 (1994)]; PO₄₁₇₅ [Haniu, M.,Iyanagi, T., Miller, P., Lee, T. D. and Shively, J. E. Complete aminoacid sequence of NADPH-cytochrome P-450 reductase from porcine hepaticmicrosomes. Biochemistry 25 (24), 7906-7911 (1986)].

[0144]FIG. 12—Phylogenetic Tree Showing the Relatedness of HumanOxidoreductases with Top 4 Hits from SwissProt

[0145] A phylogenetic tree displaying the genetic relatedness of humanoxidoreductase (SEQ ID NO: 04), cloned into plasmid pMON45604 (SEQ IDNO: 03), was aligned with related enzymes found in SWISSPROT. BLAST wasused to find the related enzymes within SWISSPROT, and ClustalW was usedgenerate the multiple sequence alignment and phylogenetic tree depictedin this figure. Descriptions of the SWISPROT accession numbers used aslabels in the figure are the same as that described above for the legendto FIG. 11, above.

[0146]FIG. 13—Percent Identity Between Human Oxidoreductase and Top 4Hits from SwissProt

[0147] The percent identity between human oxidoreductase and the top 4hits found in SWISSPROT was calculated using Clustal W and Boxshade.

[0148]FIG. 14: Expression of Aspergillus ochraceus 11 Alpha Hydroxylasein Transfected Sf9 Insect Cells

[0149] Baculovirus-infected insect cells expressing Aspergillusochraceus 11 alpha hydroxylase were harvested at 25 and 48 hours postinfection and microsomal membrane fractions were prepared and separatedby SDS-polyacrylamide gel electrophoresis. The proteins in the gel wereelectrophoretically transferred to 0.2 um nitrocellulose membrane(Schleicher & Schuell Grimsehlstrasse 23 37574 Einbeck Germany) andprobed with antibodies GN-1187 and GN-1188 prepared from peptide 11aOHpeptide 2 CRQILTPYIHKRKSLKGTTD (SEQ ID NO: 24).

[0150]FIG. 15: Expression of Aspergillus ochraceus P450 Oxidoreductasein Transfected Sf9 Insect Cells

[0151] Baculovirus-infected insect cells expressing Aspergillusochraceus 11 oxidoreductase were harvested at 25 and 48 hours postinfection and microsomal membrane fractions were prepared and separatedby SDS-polyacrylamide gel electrophoresis. The proteins in the gel wereelectrophoretically transferred to 0.2 um nitrocellulose membrane(Schleicher & Schuell Grimsehlstrasse 23 37574 Einbeck Germany) andprobed with antibodies GN-2023 and GN-12024 prepared from oxr peptide 1CTYWAVAKDPYASAGPAMNG (SEQ ID NO: 26).

[0152]FIG. 16—Conversion of Androstenedione to 11 Alpha HydroxyAndrostenedione Monitored by HPLC

[0153] Microsomal and mitochondrial subcellular fractions were preparedfrom insect cells co-infected with recombinant baculoviruses expressingrecombinant Aspergillus ochraceus 11 alpha hydroxylase and humanoxidoreductase cloned from HepG2 cell RNA. The subcellular fractionswere incubated with 250 μM androstenedione (AD) in the presence of anNADPH-generating system for 120 minutes, and the resulting products wereseparated by HPLC and monitored by ultraviolet detection at 247 nm.Hydroxlase activity was found in the microsomal fraction, as expected,but also appeared in the mitochondrial fraction. These results suggestthat the 11 alpha hydroxylase may have a tendency to stick to membranesin disrupted cells, or that the separation of the subcellular fractionsin this experiment was insufficient. Panel A illustrates a reactioncarried out using enzyme prepared from a mitochondrial fraction. Thepeak in panel A that elutes after AD appears to be testosterone. When amicrosomal fraction was used, almost as much AD was converted to 11alpha hydroxy AD, but relatively more testosterone was also produced.Panel B illustrates the same reaction carried out for 120 minuteswithout a source of enzyme. Panel C illustrates an HPLC tracing with11α-hydroxyandrostenedione standard added to incubation buffer.

DETAILED DESCRIPTION OF THE INVENTION

[0154] The present invention encompasses enzymes that facilitate thebiosynthesis of steroid molecules, particularly enzymes possessingcytochrome P450 or oxidoreductase activities. The present invention isdirected, in part, to the isolation of a nucleic acid encodingAspergillus ochraceus 11 alpha hydroxylase, which exhibits sequencehomology to the highly conserved residues that correspond to cytochromeP450 enzymes. It also directed to the isolation of nucleic acidsencoding human and Aspergillus ochraceus oxidoreductase. Biologicalactivities of the cloned hydroxylases and oxidoreductases of the presentinvention can be determined by a variety of assays, including incubationof steroid substrates in the presence of microsomes prepared fromrecombinant baculovirus-infected insect cells and monitoring theconversion to their 11 alpha hydroxy-counterparts by high pressureliquid chromatography (HPLC). The present invention, comprising novel 11alpha hydroxylase and oxidoreductase nucleic acids, proteins, peptides,homologues, and fragments of either, provides new and advantageousmethods to convert steroid intermediates to their 11 alpha hydroxycounterparts.

[0155] The present invention also includes the DNA sequences which codefor the 11 alpha hydroxylases and oxidoreductases, DNA sequences whichare substantially similar and perform substantially the same function,and DNA sequences which differ from the DNAs encoding the hydroxylasesand oxidoreductases of the invention only due to the degeneracy of thegenetic code. Also included in the present invention are theoligonucleotide intermediates used to construct mutated versions ofthese DNAs and the polypeptides encoded by these oligonucleotides andmutant DNAs.

[0156] The present invention also includes antibodies which bindspecifically to A. ochraceus 11 alpha hydroxylase or A. ochraceusoxidoreductase, including anti-peptide antibodies, methods of usingthese anti-peptide antibodies to purify these and other relatedpolypeptides, methods of using the purified polypeptides to generatepolyclonal or monoclonal antibodies to the full-length polypeptides, andmethods of using antibodies to the full-length polypeptides to assessthe presence of the polypeptides in recombinant and non-recombinant hostcells. The antibodies can be used to identify related polypeptides inany of a variety of host organisms that possess the biologicalactivities associated with these polypeptides.

[0157] Among the preferred organisms that can be used in thishydroxylation step are Aspergillus ochraceus NRRL 405, Aspergillusochraceus ATCC 18500, Aspergillus niger ATCC 16888 and ATCC 26693,Aspergillus nidulans ATCC 11267, Rhizopus oryzae ATCC 11145, Rhizopusstolonifer ATCC 6227b, Streptomyces fradiae ATCC 10745, Bacillusmegaterium ATCC 14945, Pseudomonas cruciviae ATCC 13262, andTrichothecium roseum ATCC 12543. Other preferred organisms includeFusarium oxysporum f. sp. cepae ATCC 11171 and Rhizopus arrhizus ATCC11145.

[0158] Other organisms that have exhibited activity for this reactioninclude Absidia coerula ATCC 6647, Absidia glauca ATCC 22752,Actinomucor elegans ATCC 6476, Aspergillus flavipes ATCC 1030,Aspergillus fumigatus ATCC 26934, Beauveria bassiana ATCC 7159 and ATCC13144, Botryosphaeria obtusa IMI 038560, Calonectria decora ATCC 14767,Chaetomium cochliodes ATCC 10195, Corynespora cassiicola ATCC 16718,Cunninghamella blakesleeana ATCC 8688a, Cunninghamella echinulata ATCC3655, Cunninghamella elegans ATCC 9245, Curvularia clavata ATCC 22921,Curvularia lunata ACTT 12071, Cylindrocarpon radicicola ATCC 1011,Epicoccum humicola ATCC 12722, Gongronella butleri ATCC 22822, Hypomyceschrysospermus, Mortierella isabellina ATCC 42613, Mucor mucedo ATCC4605, Mucor griseocyanus ATCC 1207A, Myrothecium verrucaria ATCC 9095,Nocardia corallina, Paecilomyces carneus ATCC 46579, Penicillum patulumATCC 24550, Pithomyces atroolivaceus IFO 6651, Pithomyces cynodontisATCC 26150, Pycnosporium sp. ATCC 12231, Saccharopolyspora erythrae ATCC11635, Sepedonium chrysospermum ATCC 13378, Stachylidium bicolor ATCC12672, Streptomyces hyqroscopicus ATCC 27438, Streptomyces purpurascensATCC 25489, Syncephalastrum racemosum ATCC 18192, Thamnostylum piriformeATCC 8992, Thielavia terricola ATCC 13807, and Verticillium theobromaeATCC 12474.

[0159] Additional organisms that may be expected to show activity forthe 11α hydroxylation include Cephalosporium aphidicola (Phytochemistry(1996), 42(2), 411-415), Cochliobolus lunatas (J. Biotechnol. (1995),42(2), 145-150), Tieghemella orchidis (Khim.-Farm.Zh. (1986), 20(7),871-876), Tieghemella hyalospora (Khim.-Farm.Zh. (1986), 20(7),871-876), Monosporium olivaceum (Acta Microbiol. Pol., Ser. B. (1973),5(2), 103-110), Aspergillus ustus (Acta Microbiol. Pol., Ser. B. (1973),5(2), 103-110), Fusarium graminearum (Acta Microbiol. Pol., Ser. B.(1973), 5(2), 103-110), Verticillium glaucum (Acta Microbiol. Pol., Ser.B. (1973), 5(2), 103-110), and Rhizopus nigricans (J. Steroid Biochem.(1987), 28(2), 197-201).

[0160]FIG. 1 sets forth the nucleotide and protein sequence ofAspergillus ochraceus 11 alpha hydroxylase (SEQ ID NO: 1, SEQ ID NO: 2,respectively). FIG. 2 sets forth the nucleotide and protein sequence ofhuman oxidoreductase (SEQ ID NO: 3, SEQ ID NO: 4, respectively). FIG. 3sets forth the nucleotide and protein sequence of Aspergillus ochraceusoxidoreductase (SEQ ID NO: 5, SEQ ID NO: 6, respectively).

[0161]FIG. 4 sets forth an amino acid homology alignment of A. ochraceus11 alpha hydroxylase cloned in pMON45624 and aligned with relatedenzymes found in GenBank using BLAST. FIG. 5 is a phylogenetic treeshowing the this relationship graphically. FIG. 6 shows the percenthomology between Aspergillus ochraceus steroid 11 alpha hydroxylase andthe top 10 enzymes found in GenBank using BLAST, calculated usingClustal W and Boxshade.

[0162]FIG. 7 sets forth the amino acid homology of Aspergillus ochraceusand human oxidoreductase to NADPH cytochrome P450 reductases from A.niger, mouse, and S. cerevisiae (yeast). FIG. 8 sets forth the aminoacid alignment for A. ochraceus, A. niger, and S. cerevisiaeoxidoreductases. FIG. 9 is a phylogenetic tree showing the relatednessof Aspergillus ochraceus and human oxidoreductase to reductases from A.niger, yeast, and mouse. FIG. 10 shows the percent homology betweenAspergillus ochraceus steroid 11 alpha hydroxylase and theoxidoreductases from A. niger, yeast, and mouse, calculated usingClustal W and Boxshade.

[0163]FIG. 11—Alignment of human oxidoreductase with top 4 hits fromSwissProt. FIG. 12 sets forth a phylogenetic tree displaying the geneticrelatedness of human oxidoreductase, to these hits. FIG. 13 shows thepercent identity between human oxidoreductase and top 4 hits fromSwissProt.

[0164]FIG. 14 sets forth an immunoblot illustrating expression ofAspergillus ochraceus P450 11 alpha hydroxylase in baculovirus-infectedinsect cells harvested at 25 and 48 hours post infection. Thenitrocellulose membrane was probed with a 1:1 mixture of antibodiesprepared from two rabbits immunized with a conjugated synthetic peptide,11aOH peptide 2 (SEQ ID NO 24).

[0165]FIG. 15 sets forth an immunoblot illustrating expression ofAspergillus ochraceus P450 oxidoreductase in baculovirus-infected insectcells harvested at 25 and 48 hours post infection. The nitrocellulosemembrane was probed with a 1:1 mixture of antibodies prepared tworabbits immunized with a conjugated synthetic peptide, oxr peptide 1(SEQ ID NO 26).

[0166]FIG. 16 sets forth an HPLC tracing illustrating the conversion ofandrostenedione (AD) to its 11 alpha hydroxy counterpart afterincubating AD with subcellular fractions prepared frombaculovirus-infected insect cells expressing Aspergillus ochraceus 11alpha hydroxylase and human oxidoreductase.

[0167] Cloning Techniques

[0168] Genetic engineering techniques now standard in the art (U.S. Pat.No. 4,935,233 and Sambrook et al., “Molecular Cloning A LaboratoryManual”, Cold Spring Harbor Laboratory, 1989) may be used in theconstruction of the DNA sequences of the present invention. One suchmethod is cassette mutagenesis (Wells et al., Gene 34:315-323, 1985) inwhich a portion of the coding sequence in a plasmid is replaced withsynthetic oligonucleotides that encode the desired amino acidsubstitutions in a portion of the gene between two restriction sites.

[0169] Pairs of complementary synthetic oligonucleotides encoding thedesired gene can be made and annealed to each other. The DNA sequence ofthe oligonucleotide would encode sequence for amino acids of desiredgene with the exception of those substituted and/or deleted from thesequence.

[0170] Plasmid DNA can be treated with the chosen restrictionendonucleases then ligated to the annealed oligonucleotides. The ligatedmixtures can be used to transform competent E. coli cells which willconfer resistance to an appropriate antibiotic. Single colonies can bepicked and the plasmid DNA examined by restriction analysis or by DNAsequencing to identify plasmids with the desired genes.

[0171] Cloning of DNA sequences encoding novel proteins and fusionproteins may be accomplished by the use of intermediate vectors. Linkersand adapters can be used to join DNA sequences, and to replace lostsequences, where a restriction site is internal to the region ofinterest. DNA encoding a single polypeptide or a fusion protein(comprising a first polypeptide, a peptide linker, and a secondpolypeptide) is inserted into a suitable expression vector which is thentransformed or transfected into appropriate bacterial, fungal, insect,or mammalian host cells. The transformed organism or host cell line isgrown and the recombinant protein isolated by standard techniques.Recombinant fusion proteins have all or a portion of a first proteinjoined by a linker region to a all or a portion of second protein.

[0172] Hybridization

[0173] Nucleic acid molecules and fragment nucleic acid moleculesencoding 11 alpha hydroxylases or oxidoreductases can specificallyhybridize with other nucleic acid molecules. Two nucleic acid moleculesare said to be capable of specifically hybridizing to one another if thetwo molecules are capable of forming an anti-parallel, double-strandednucleic acid structure. A nucleic acid molecule is said to be the“complement” of another nucleic acid molecule, if they exhibit completecomplementarity. Molecules exhibit complete “complementarity” when everynucleotide of one of the molecules is complementary to a nucleotide ofthe other. Two molecules are “minimally complementary” if they canhybridize to one another with sufficient stability to permit them toremain annealed to one another under at least conventional“low-stringency” conditions. Similarly, the molecules are“complementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another underconventional “high-stringency” conditions. Conventional stringencyconditions are described by Sambrook, et al., Molecular Cloning, ALaboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1989), and by Haymes, et al. Nucleic Acid Hybridization, APractical Approach, IRL Press, Washington, D.C., 1985). Departures fromcomplete complementarity are therefore permissible, as long as suchdepartures do not completely preclude the capacity of the molecules toform a double-stranded structure.

[0174] Appropriate stringency conditions which promote DNA hybridizationare well known to those skilled in the art, or can be found in CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y., 6.3.1-6.3.6,(1989). Basic conditions would include, for example, 6×sodium salinecitrate (SSC) at about 45° C., followed by a wash of 2×SSC at 50° C.Stringency can be varied, for example, by altering the saltconcentration in the wash step from about 2×SSC at 50° C. (moderatelylow stringency) to about 0.2×SSC at 50° C. (high stringency). Stringencycan also be altered by changing the temperature in the wash step, fromroom temperature, about 22° C. (low stringency conditions), to about 65°C. (high stringency conditions). Both temperature and salt may bevaried, or either the temperature or the salt concentration may be heldconstant while the other variable is changed.

[0175] Expression Vectors

[0176] Another aspect of the present invention includes plasmid DNAvectors for use in the expression of these novel hydroxylases andoxidoreductases. These vectors contain the novel DNA sequences describedabove which code for the novel polypeptides of the invention.Appropriate vectors which can transform microorganisms or cell linescapable of expressing the hydroxylases and oxidoreductases includeexpression vectors comprising nucleotide sequences coding for thehydroxylases and oxidoreductases joined to transcriptional andtranslational regulatory sequences which are selected according to thehost cells used.

[0177] Vectors incorporating modified sequences as described above areincluded in the present invention and are useful in the production ofthe hydroxylases and oxidoreductases. The vector employed in the methodalso contains selected regulatory sequences in operative associationwith the DNA coding sequences of the invention and which are capable ofdirecting the replication and expression thereof in selected host cells.

[0178] Methods for producing the hydroxylases and oxidoreductases isanother aspect of the present invention. The method of the presentinvention involves culturing suitable cells or cell lines, which hasbeen transformed with a vector containing a DNA sequence encoding novelhydroxylases and oxidoreductases. Suitable cells or cell lines may bebacterial cells. For example, various strains of E. coli are well-knownas host cells in the field of biotechnology. Examples of such strainsinclude E. coli strains DH5 alpha, DH10B and MON105 (Obukowicz et al.,Applied Environmental Microbiology 58: 1511-1523, 1992). Also includedin the present invention is the expression of the hydroxylases andoxidoreductases utilizing a chromosomal expression vector for E. colibased on the bacteriophage Mu (Weinberg et al., Gene 126: 25-33, 1993).Various other strains of bacteria, including the Enteric bacteria (e.g.,Salmonella sp.) and B. subtilis, may also be employed in this method.

[0179] When expressed in the E. coli cytoplasm, the gene encoding theproteins of the present invention may also be constructed such that atthe 5′ end of the gene codons are added to encode Met⁻²—Ala⁻¹,Met⁻²—Ser⁻¹, Met⁻²—Cys⁻¹, or Met⁻¹ at the N-terminus of the protein. TheN termini of proteins made in the cytoplasm of E. coli are affected bypost-translational processing by methionine aminopeptidase (Ben Bassatet al., J. Bacteriol. 169:751-757, 1987), and possibly by otherpeptidases, so that upon expression the methionine is cleaved off theN-terminus. The proteins of the present invention may includepolypeptides having Met⁻¹, Ala⁻¹, Ser⁻¹, Cys⁻¹, Met⁻²—Ala⁻¹,Met⁻²—Ser⁻¹, or Met⁻²—Cys⁻¹ at the N-terminus. These mutant proteins mayalso be expressed in E. coli by fusing a secretion signal peptide to theN-terminus. This signal peptide is cleaved from the polypeptide as partof the secretion process.

[0180] Yeast

[0181] Many strains of yeast cells known to those skilled in the art arealso available as host cells for expression of the polypeptides of thepresent invention. Under another embodiment, the protein or fragmentthereof of the present invention is expressed in a yeast cell,preferably Saccharomyces cerevisiae. The proteins or fragments thereofof the present invention can be expressed in S. cerevisiae by fusing itto the N-terminus of the URA3, CYC1 or ARG3 genes (Guarente and Ptashne,Proc. Natl. Acad. Sci. (U.S.A.) 78:2199-2203 (1981); Rose et al., Proc.Natl. Acad. Sci. (U.S.A.) 78:2460-2464 (1981); and Crabeel et al., EMBOJ. 2:205-212 (1983)). Alternatively, proteins or fragments thereof ofthe present invention can be fused to either the PGK or TRP1 genes(Tuite et al., EMBO J. 1:603-608 (1982); and Dobson et al., NucleicAcids. Res. 11:2287-2302 (1983)). More preferably, the protein orfragment thereof of the present invention is expressed as a matureprotein (Hitzeman et al., Nature 293:717-722 (1981); Valenzuela et al.,Nature 298:347-350 (1982); and Derynck et al., Nucleic Acids Res.11:1819-1837 (1983)).

[0182] Native and engineered yeast promoters suitable for use in thepresent invention have been reviewed by Romanos et al., Yeast 8:423-488(1992). Most preferably, the protein or fragment thereof of the presentinvention is secreted by the yeast cell (Blobel and Dobberstein, J. CellBiol. 67:835-851 (1975); Kurjan and Herskowitz, Cell 30:933-943 (1982);Bostian et al., Cell 36:741-751 (1984); Rothman and Orci, Nature355:409-415 (1992); Julius et al., Cell 32:839-852 (1983); and Julius etal., Cell 36:309-318 (1984)).

[0183] Mammalian

[0184] General methods for expression of foreign genes in mammaliancells have been reviewed (Kaufman, R. J., 1987, “Genetic Engineering,Principles and Methods”, Vol. 9, J. K. Setlow, editor, Plenum Press, NewYork; Colosimo et al., Biotechniques 29: 314-331, 2000). Recombinantproteins are generally targeted to their natural locations within thehost cell (e.g., cytoplasm, nucleus, or various membrane compartments),or are secreted, if a signal peptide is present. An expression vector isconstructed in which a strong promoter capable of functioning inmammalian cells drives transcription of a eukaryotic secretion signalpeptide coding region, which is translationally joined to the codingregion for the desired protein. For example, plasmids such as pcDNAI/Neo, pRc/RSV, and pRc/CMV (obtained from Invitrogen Corp., San Diego,Calif.) can be used. The eukaryotic secretion signal peptide codingregion can be from the gene itself or it can be from another secretedmammalian protein (Bayne, M. L. et al., Proc. Natl. Acad. Sci. USA 84:2638-2642, 1987). After construction of the vector containing the gene,the vector DNA is transfected into mammalian cells such as the COS7,HeLa, BHK, Chinese hamster ovary (CHO), or mouse L lines. The cells canbe cultured, for example, in DMEM media (JRH Scientific). Thepolypeptide secreted into the media can be recovered by standardbiochemical approaches following transient expression for 24-72 hoursafter transfection of the cells or after establishment of stable celllines following selection for antibiotic resistance. The selection ofsuitable mammalian host cells and methods for transformation, culture,amplification, screening and product production and purification areknown in the art. See, e.g., Gething and Sambrook, Nature, 293:620-625,1981, or alternatively, Kaufman et al, Mol. Cell. Biol., 5(7):1750-1759,1985) or Howley et al., and U.S. Pat. No. 4,419,446. Other suitablemammalian cell lines are the monkey COS-1 cell line and the CV-1 cellline.

[0185] Mammalian cells can also be used to express the nucleic acidmolecules of the present invention. The nucleic acid molecules of thepresent invention can be cloned into a suitable retroviral vector (see,e.g., Dunbar et al., Blood 85:3048-3057 (1995); Baum et al., J.Hematother. 5: 323-329 (1996); Bregni et al., Blood 80:1418-1422 (1992);Boris-Lawrie and Temin, Curr. Opin. Genet. Dev. 3:102-109 (1993);Boris-Lawrie and Temin, Annal. New York Acad. Sci. 716:59-71 (1994);Miller, Current Top. Microbiol. Immunol. 158:1-24 (1992)), adenovirusvector (Berkner, BioTechniques 6:616-629 (1988); Berkner, Current Top.Microbiol. Immunol. 158:39-66 (1992); Brody and Crystal, Annal. New YorkAcad. Sci. 716:90-103 (1994); Baldwin et al., Gene Ther. 4:1142-1149(1997)), RSV, MuSV, SSV, MuLV (Baum et al., J. Hematother. 5: 323-329(1996)), AAV (Chen et al., Gene Ther. 5:50-58 (1998); Hallek et al.,Cytokines Mol. Ther. 2: 69-79 (1996)), AEV, AMV, or CMV (Griffiths etal., Biochem. J. 241: 313-324 (1987)).

[0186] Transformation and Transfection

[0187] In another aspect, the invention provides a transformed cellhaving a nucleic acid molecule which comprises an exogenous promoterregion which functions in a cell to cause the production of an mRNAmolecule which is linked to a structural nucleic acid molecule, whereinthe structural nucleic acid molecule encodes an 11 alpha hydroxylase oroxidoreductase gene or fragment thereof. This nucleic acid molecule islinked to a 3′ non-translated sequence that functions in a cell to causetermination of transcription and addition of polyadenylatedribonucleotides to a 3′ end of the mRNA molecule.

[0188] Methods and compositions for transforming eukaryotic cells,bacteria and other microorganisms are known in the art (see, forexample, Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989); Colosimo et al., Biotechniques 29: 314-331, 2000).

[0189] Technology for introduction of DNA into cells is well known tothose of skill in the art. Four general methods for delivering a geneinto cells have been described: (1) chemical methods (Graham and van derEb, Virology 54:536-539 (1973)); (2) physical methods such asmicroinjection (Capecchi, Cell 22:479-488 (1980)), electroporation (Wongand Neumann, Biochem. Biophys. Res. Commun. 107:584-587 (1982); Fromm etal., Proc. Natl. Acad. Sci. (U.S.A.) 82:5824-5828 (1985); U.S. Pat. No.5,384,253); and the gene gun (Johnston and Tang, Methods Cell Biol.43:353-365 (1994); (3) viral vectors (Clapp, Clin. Perinatol. 20:155-168(1993); Lu et al., J. Exp. Med. 178:2089-2096 (1993); Eglitis andAnderson, Biotechniques, 6:608-614 (1988)); and (4) receptor-mediatedmechanisms (Curiel et al., Hum. Gen. Ther. 3:147-154 (1992), Wagner etal., Proc. Natl. Acad. Sci. (U.S.A.) 89:6099-6103 (1992)). Other methodswell known in the art can also be used.

[0190] Transformation can be achieved using methods based on calciumphosphate precipitation, polyethylene glycol treatment, electroporation,and combinations of these treatments (see for example Potrykus et al.,Mol. Gen. Genet. 205:193-200 (1986); Lorz et al., Mol. Gen. Genet.199:178 (1985); Fromm et al., Nature 319:791 (1986); Uchimiya et al.,Mol. Gen. Genet. 204:204 (1986); Marcotte et al., Nature 335:454-457(1988)).

[0191] Assays for gene expression based on the transient expression ofcloned nucleic acid constructs have been developed by introducing thenucleic acid molecules into cells by polyethylene glycol treatment,electroporation, or particle bombardment (Marcotte et al., Nature 335:454-457 (1988); McCarty et al., Cell 66: 895-905 (1991); Hattori et al.,Genes Dev. 6: 609-618 (1992); Goffet al., EMBO J. 9: 2517-2522 (1990)).Transient expression systems may be used to functionally dissect theregulatory and structural features of expression cassettes comprisingoperably-linked genetic elements.

[0192] Insect Cell Expression

[0193] Insect cells may be used as host cells to express recombinantproteins of the present invention (See, e.g., Luckow, V. A., ProteinEng. J. L. Cleland., Wiley-Liss, New York, N.Y.: 183-218, 1996, andreferences cited therein). General methods for expression of foreigngenes in insect cells using baculovirus vectors have been described(O'Reilly, D. R., L. K. Miller et al. Baculovirus Expression Vectors: ALaboratory Manual. New York, W. H. Freeman and Company, 1992; and King,L. A. and R. D. Possee, The Baculovirus Expression System: A LaboratoryGuide, London, Chapman & Hall).

[0194] A baculovirus expression vector can be constructed by insertingthe desired gene (e.g., 11 alpha hydroxylase or oxidoreductase) into abaculovirus transfer vector which can recombine into the baculovirusgenome by homologous recombination. Many transfer vectors use a strongbaculovirus promoter (such as the polyhedrin promoter) to drivetranscription of the desired gene. Some vectors permit the expression offusion proteins or direct the secretion of proteins from the cell byfusing a eukaryotic secretion signal peptide coding region to the codingregion of the desired gene. The plasmid pVL1393 (obtained fromInvitrogen Corp., San Diego, Calif.) can be used, for example, to directtranscription of nonfused foreign genes in baculovirus-infected insectcells. The baculovirus transfer vector containing the desired gene istransfected into Spodoptera frugiperda (Sf9) insect cells along withcircular or linearized genomic baculovirus DNA, and recombinantbaculoviruses purified and amplified after one or more plaque assays.

[0195] Recombinant baculoviruses can also be created using thebaculovirus shuttle vector system (Luckow, V. A. et al., J. Virol.67(8): 4566-4579, 1993; U.S. Pat. No. 5,348,886) now marketed as theBac-To-Bac™ Expression System (Life Technologies, Inc., Rockville, Md.).The desired genes are inserted downstream from the polyhedrin promoterin mini-Tn7 cassettes that are transposed in vivo into a baculovirusshuttle vector genome propagated in E. coli. Composite viral DNAs areisolated from E. coli and transfected into Sf9 cells and stocks ofrecombinant baculoviruses are rapidly prepared without the need formultiple rounds of tedious plaque purification common to methods thatrely on homologous recombination.

[0196] Recombinant baculoviruses can also created using the GatewayRecombinational Cloning System (Life Technologies) of shuttling genesfrom vector to vector using modified genetic elements (attachment sites)and modified proteins (e.g., int, IHF, xis) that are involved in thesite-specific integration and excision of bacteriophage lambda.

[0197] Pure recombinant baculoviruses carrying the 11 alpha hydroxylaseor oxidoreductase gene are used to infect cells cultured, for example,in Excell 401 serum-free medium (JRH Biosciences, Lenexa, Kans.) orSf900-II (Life Technologies). Hydroxylases or oxidoreductases that arelocalized to membranes can be prepared using standard protocols thatfractionate and enrich for enzymes in mitochondrial or microsomalfractions (Engel and White, Dev Biol. 140: 196-208, 1990). Hydroxylasesor oxidoreductases that are secreted or leak into the medium can also berecovered by standard biochemical approaches.

[0198] Simultaneous expression of two or more recombinant proteins inbaculovirus-infected insect cells can be carried out by two generalapproaches. The simplest approach is to coinfect insect cells withtitered stocks of recombinant baculoviruses harboring a singleheterologous gene under the control of a strong baculovirus promoter,such as the polyhedrin or the plO promoter. These promoters are highlytranscribed during the late stages of infection when most host cellprotein synthesis has been shut down. Earlier baculovirus promoters orother insect or eukaryotic cell promoters can also be used to directsynthesis at other times, which generally result in lower expressionlevels. Varying the ratio of two or more recombinant viruses used in acoinfection or selecting viruses that use different promoters to driveexpression of the recombinant protein will permit one skilled in the artto select conditions suitable for optimal expression of the desiredrecombinant proteins.

[0199] Construction of dual- or multiple-expression vectors will alsopermit the expression of two or more recombinant proteins inbaculovirus-infected insect cells. Generally, these vectors permit theintroduction two or more gene cassettes into a single locus in thebaculovirus genome. The structures of a variety of dual expressionvectors have been described (O'Reilly, D. R., L. K. Miller et al.Baculovirus Expression Vectors: A Laboratory Manual. New York, W. H.Freeman and Company, 1992; and King, L. A. and R. D. Possee, TheBaculovirus Expression System: A Laboratory Guide, London, Chapman &Hall).

[0200] Materials and Methods

[0201] General Methods

[0202] General methods of cloning, expressing, and characterizingproteins are found in T. Maniatis, et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, 1982, and referencescited therein, incorporated herein by reference; and in J. Sambrook, etal., Molecular Cloning, A Laboratory Manual, 2^(nd) edition, Cold SpringHarbor Laboratory, 1989, and references cited therein, incorporatedherein by reference. General features and maps of a wide variety ofcloning and expression vectors have been also been published (Gacesa, P.and Ramji, D. P., Vectors: Essential Data, John Wiley & Sons, 1994).General methods for the cloning and expression of genes in mammaliancells are also found in Colosimo et al., Biotechniques 29: 314-331,2000. General and specific conditions and procedures for theconstruction, manipulation and isolation of polyclonal and monoclonalantibodies are well known in the art (See, for example, Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Press, Cold SpringHarbor, N.Y., 1988).

[0203] Unless noted otherwise, all specialty chemicals were obtainedfrom Sigma (St. Louis, Mo.). Restriction endonucleases and T4 DNA ligasewere obtained from Life Technologies (Rockville, Md.), New EnglandBiolabs (Beverly, Mass.), Roche Molecular Biochemicals (Indianapolis,Ind.), or Promega (Madison, Wis.). All parts are by weight andtemperatures are in degrees centigrade (° C.), unless otherwiseindicated.

[0204] Strains, Plasmids, and Sequence Cross Listings

[0205] The bacterial strains used in these studies are listed inTable 1. Plasmids used or constructed for this study are listed in Table2. Brief descriptions of sequences of relevant oligonucleotides, genes,or proteins are listed in Table 3. TABLE 1 Strains DesignationDescription or Genotype Reference/Source DH5α ™ F, phi80 dlacZdeltaM15,Life Technologies, Rockville, delta(lacZYA-argF)U169, deoR, recA1,Maryland endA1, hsdR17 (rk⁻, mk⁺), phoA, supE44, lambda-, thi-1, gyrA96,relA1 DH10B ™ F, mcrA D(mrr-hsdRMS-mcrBC) Life Technologies, Rockville,phi80 dlacZDM15 DlacX74 endA1 Maryland recA1 deoR D(ara, leu)7697araD139 galU galK nupG rpsL DH10Bac ™ DH10B harboring the baculovirusLife Technologies, Rockville, shuttle vector bMON14272 (Kan^(R))Maryland; See also Luckow et al., J. and the helper plasmid pMON7124Virol. 67: 4566-4579 (1993) (Tet^(R))

[0206] TABLE 2 Plasmids Plasmid SEQ ID NO. Marker Description SourcepFastBac1 Amp^(R) Baculovirus donor plasmid containing Life TechnologiesGent^(R) multiple cloning site downstream Inc. (Rockville, of an AcNPVpolyhedrin promoter MD); See also within a mini-Tn7 transposable Luckowet al., element capable of being transposed J. Virol. 67: to abaculovirus shuttle vector 4566-4579 (1993) pBluescript II Amp^(R)Multifunctional phagemid cloning Stratagene, La SK vector derived frompUC19. Jolla, CA pCRII-TOPO Amp^(R) Multifunctional cloning vectorInvitrogen, Kan^(R) for direct cloning of polymerase Carlsbad, CA chainreaction products using the T overhang pSport1 Amp^(R) Multifunctionalcloning vector for Life Technologies, cloning and in vitro transcriptionRockville, MD from either strand using SP6 or T7 promoters pGEM-TAmp^(R) A derivative of pGEM-5Zf(+) with Promega, single 5' T overhangsat the Madison, WI insertion site to improve the efficiency of PCRproduct ligation pMON45624 #1 Amp^(R) pFastBac1 EcoRI/XbaI + PCR Thiswork Gent^(R) fragment EcoRI/XbaI encoding Aspergillus ochraceus 11alpha hydroxylase pMON45603 Amp^(R) pBluescriptII SK BamHI/HincII + Thiswork BamHI/HincII 5' segment of human oxidoreductase pMON45604 Amp^(R)pBluescriptII SK HincII/KpnI + This work HincII/KpnI 3' segment of humanoxidoreductase pMON45605 #3 Amp^(R) pFastBac1 BamHI/KpnI + BamHI/ Thiswork Gent^(R) KpnI complete coding region of human oxidoreductase cDNA.pMON45630 Amp^(R) pCRII-TOPO SalI/BamHI + SalI/ This work Kan^(R) BamHI5' segment of A. ochraceus oxidoreductase cDNA pMON45631 Amp^(R)pCRII-TOPO BamHI/XhoI + This work Kan^(R) BamHI/XhoI 3' segment of A.ochraceus oxidoreductase cDNA which lacked the intron. pMON45632 #5Amp^(R) pFastBac1 SalI/XhoI + containing This work Gent^(R) assembledcoding region of Aspergillus ochraceus oxidoreductase

[0207] TABLE 3 Table of Sequences SEQ ID NO Description Length/SequenceType (SEQ ID NO: 01) Nucleotide sequence of 1776 DNA Aspergillusochraceus 11alphaOH gene from pMON45624 (SEQ ID NO: 02) Aspergillusochraceus  514 Protein 11alphaOH protein sequence from pMON45624 (SEQ IDNO: 03) Nucleotide sequence of human 2031 DNA oxidoreductase gene frompMON45605 (SEQ ID NO: 04) Human oxidoreductase protein  677 Proteinsequence from pMON45605 (SEQ ID NO: 05) Nucleotide sequence of 2322 DNAAspergillus ochraceus oxidoreductase gene from pMON45632 (SEQ ID NO: 06)Aspergillus ochraceus  705 Protein oxidoreductase protein sequence frompMON45632 (SEQ ID NO: 07) Primer H. oxred 1A gatcggatccaatATGG DNAGAGACTCCCACGTGGAC AC (SEQ ID NO: 08) Primer H. oxred 1BCAGCTGGTTGACGAGAG DNA CAGAG (SEQ ID NO: 09) Primer H. oxred 2ACTCTGCTCTCGTCAACC DNA AGCTG (SEQ ID NO: 10) Primer H. oxred 2BgatcggtaccttaGCTC DNA CACACGTCCAGGGAGTA G (SEQ ID NO: 11) PrimerA.oxred-for1 GACGGIGCIGGTACAAT DNA GCA (SEQ ID NO: 12) PrimerA.oxred-rev1 TTAIGACCAIACATCIT DNA CCTGGTAGC (SEQ ID NO: 13) PrimerpSport-for1 CAAGCTCTAATACGACT DNA CACTATAGGGA (SEQ ID NO: 14) PrimerA.oxred-rev2 CAGGAACCGATCGACCT DNA CGGAA (SEQ ID NO: 15) PrimerA.oxred-rev3 GTCACCCTCACCAGCAG DNA AGCCAATG (SEQ ID NO: 16) PrimerA.oxred-rev4 CCACATTGCGAACCATA DNA GCGTTGTAGTG (SEQ ID NO: 17) PrimerpSport-for2 GCCAAGCTCTAATACGA DNA CTCACTATAGGGAAAGC (SEQ ID NO: 18)Primer A.oxred-for2 gtcgacATGGCGCAACT DNA CGATACTCTC (SEQ ID NO: 19)Primer A.oxred-rev5 ctcgagttaGGACCAGA DNA CATCGTCCTGGTAG (SEQ ID NO: 20)Primer A.oxred-for3 GGATCCCTCGCGACCTG DNA TGATCAT (SEQ ID NO: 21) PrimerA.oxred-for4 CGAAGATTTCTTGTACA DNA AGGATGAATGGAAGACT TTTC (SEQ ID NO:22) Primer A.oxred-rev6 CTGAAAAGTCTTCCATT DNA CATCCTTGTACAAGAAA TC (SEQID NO: 23) 11aOH peptide 1 AAAYWLATLQPSDLPEL Protein N (SEQ ID NO: 24)11aOH peptide 2 CRQILTPYIHKRKSLKG Protein TTDE (SEQ ID NO: 25) 11aOHpeptide 3 HMGFGHGVHACPGRFFA Protein SNEI (SEQ ID NO: 26) oxr peptide 1CTYWAVAKDPYASAGPA Protein MNG (SEQ ID NO: 27) CAA75565; cytochrome P450Protein monooxygenase [Gibberella fujikuroi] (SEQ ID NO: 28) CAB91316;probable cytochrome Protein P450 monooxygenase (lovA) [Neurosporacrassa] (SEQ ID NO: 29) CAB56503; cytochrome P450 Protein [Catharanthusroseus] (SEQ ID NO: 30) AAB94588; CYP71D10p [Glycine Protein max] (SEQID NO: 31) CAA75566; cytochrome P450 Protein monooxygenase [Gibberellafujikuroi] (SEQ ID NO: 32) AAD34552; cytochrome P450 Proteinmonooxygenase [Aspergillus terreus] (SEQ ID NO: 33) CAA75567; cytochromeP450 Protein monooxygenase [Gibberella fujikuroi] (SEQ ID NO: 34)CAA76703; cytochrome P450 Protein [Gibberella fujikuroi] (SEQ ID NO: 35)CAA57874; unnamed protein Protein product [Fusarium oxysporum] (SEQ IDNO: 36) CAA91268; similar to Protein cytochrome P450˜cDNA EST yk423b11.3comes from this gene; [Caenorhabditis elegans] (SEQ ID NO: 37) BAA02936NADPH-cytochrome P450 Protein reductase precursor [Saccharomycescerevisiae] (SEQ ID NO: 38) CAA81550 NADPH cytochrome P450 Proteinoxidoreductase [Aspergillus niger] (SEQ ID NO: 39) BAA04496NADPH-cytochrome P450 Protein oxidoreductase [Mus musculus] (SEQ ID NO:40) Universal bacteriophage M13 CAG GAA ACA GCT DNA reverse primer ATGAC (SEQ ID NO: 41) Universal bacteriophage T7 TAA TAC GAC TCA DNApromoter primer CTA TAG GG (SEQ ID NO: 42) Aspergillus ochraceus PrimergatcgaattcATGCCCT DNA 11alphaOH- for TCTTCACTGGGCT (SEQ ID NO: 43)Aspergillus ochraceus Primer gatctctagattacaca DNA 11alphaOH-revgttaaactcgccaTATC GAT (SEQ ID NO: 44) pFastBac1 Primer BacfwdCTGTTTTCGTAACAGTT DNA TTG (SEQ ID NO: 45) pFastBac1 Primer PolyACCTCTACAAATGTGGTA DNA TG (SEQ ID NO: 46) Aspergillus ochraceus PrimerGAGATCAAGATTGCCTT DNA 45624-for1 (SEQ ID NO: 47) Aspergillus ochraceusPrimer CTTCGACGCTCTCAA DNA 45624-for2 (SEQ ID NO: 48) Aspergillusochraceus Primer GCAATCTTGATCTCGTT DNA 45624-rev1 (SEQ ID NO: 49) S90469human cytochrome P450 2403 DNA reductase [placental, mRNA Partial, 2403nt]. (SEQ ID NO: 50) AAB21814 human cytochrome P450  676 Proteinreductase, placental, partial (SEQ ID NO: 51) A60557 human NADPH-  677Protein ferrihemoprotein reductase (SEQ ID NO: 52) P16435 HumanNADPH-cytochrome  677 Protein P450 reductase (SEQ ID NO: 53) P00389Rabbit NADPH-cytochrome  679 Protein P450 reductase (SEQ ID NO: 54)P00388 Rat NADPH-cytochrome  678 Protein P450 reductase (SEQ ID NO: 55)P37040 Mouse NADPH-cytochrome  678 Protein P450 reductase (SEQ ID NO:56) P04175 Pig NADPH-cytochrome  678 Protein P450 reductase (SEQ ID NO:57) Universal bacteriophage SP6 gatttaggtgacactat DNA primer ag (SEQ IDNO: 58) NotI-poly-dT adapter 5′- pGACTAGT DNA TCTAGA TCGCGA GCGGCCGC CC(T)₁₅ -3′ (SEQ ID NO: 59) SalI adapter, top strand 5′- DNATCGACCCACGCGTCCG -3′ (SEQ ID NO: 60) SalI adapter, bottom strand 3′- DNAGGGTGCGCAGGCp- 5′ (SEQ ID NO: 61) Primer oxred 1C GTGGACCACAAGCTCGT DNAACTG (SEQ ID NO: 62) Primer oxred 2C CATCGACCACCTGTGTG DNA AGCTG (SEQ IDNO: 63) Primer oxred 2D GTACAGGTAGTCCTCAT DNA CCGAG (SEQ ID NO: 64)Aspergillus niger NADP CYP450 3710 DNA oxidoreductase Z26838 (SEQ ID NO:65) Aspergillus niger NADP CYP450  693 Protein oxidoreductase CAA81550

[0208] Specific Methods

[0209] Transformation of E. coli Strains

[0210]E. coli strains such as DH5 alpha and DH10B (Life Technologies,Rockville, Md.) are routinely used for transformation of ligationreactions and are the hosts used to prepare plasmid DNA for transfectingmammalian cells. E. coli strains, such as DH10B and MON105 (Obukowicz,et al., Appl. and Envir. Micr., 58: 1511-1523, 1992) can be used forexpressing the proteins of the present invention in the cytoplasm orperiplasmic space.

[0211] DH10B and DH5alpha subcloning efficiency cells are purchased ascompetent cells and are ready for transformation using themanufacturer's protocol. Other E. coli strains are rendered competent totake up DNA using a CaCl₂ method. Typically, 20 to 50 mL of cells aregrown in LB medium (1% Bacto-tryptone, 0.5% Bacto-yeast extract, 150 mMNaCl) to a density of approximately 1.0 absorbance unit at 600nanometers (OD600) as measured by a Baush & Lomb Spectronicspectrophotometer (Rochester, N.Y.). The cells are collected bycentrifugation and resuspended in one-fifth culture volume of CaCl₂solution [50 mM CaCl₂, 10 mM Tris-Cl ((10 mM 2-amino-2-(hydroxymethyl)1,3-propanediol hydrochloride, pH 7.4] and are held at 4° C. for 30minutes. The cells are again collected by centrifugation and resuspendedin one-tenth culture volume of CaCl₂ solution. Ligated DNA is added to0.1 ml of these cells, and the samples are held at 4° C. for 30-60minutes. The samples are shifted to 42° C. for 45 seconds and 1.0 ml ofLB is added prior to shaking the samples at 37° C. for one hour. Cellsfrom these samples are spread on plates (LB medium plus 1.5% Bacto-agar)containing either ampicillin (100 micrograms/mL, ug/ml) when selectingfor ampicillin-resistant transformants, or spectinomycin (75 ug/ml) whenselecting for spectinomycin-resistant transformants. The plates areincubated overnight at 37° C. Colonies are picked and inoculated into LBplus appropriate antibiotic (100 ug/ml ampicillin or 75 ug/mlspectinomycin) and are grown at 37° C. while shaking.

[0212] DNA Isolation and Characterization

[0213] Plasmid DNA can be isolated by a number of different methods andusing commercially available kits known to those skilled in the art.Plasmid DNA is isolated using the Promega Wizard™ Miniprep kit (Madison,Wis.), the Qiagen QIAwell Plasmid isolation kits (Chatsworth, Calif.) orQiagen Plasmid Midi or Mini kit. These kits follow the same generalprocedure for plasmid DNA isolation. Briefly, cells are pelleted bycentrifugation (5000×g), the plasmid DNA released with sequentialNaOH/acid treatment, and cellular debris is removed by centrifugation(10000×g). The supernatant (containing the plasmid DNA) is loaded onto acolumn containing a DNA-binding resin, the column is washed, and plasmidDNA eluted. Mter screening for the colonies with the plasmid ofinterest, the E. coli cells are inoculated into 50-100 ml of LB plusappropriate antibiotic for overnight growth at 37° C. in an airincubator while shaking. The purified plasmid DNA is used for DNAsequencing, further restriction enzyme digestion, additional subcloningof DNA fragments and transfection into E. coli, mammalian cells, orother cell types.

[0214] DNA Sequencing Protocols

[0215] Purified plasmid DNA is resuspended in dH₂O and its concentrationis determined by measuring the absorbance at 260/280 nm in a Baush andLomb Spectronic 601 UV spectrometer. DNA samples are sequenced using ABIPRISM™ DyeDeoxy™ terminator sequencing chemistry (Applied BiosystemsDivision of Perkin Elmer Corporation, Lincoln City, Calif.) kits (PartNumber 401388 or 402078) according to the manufacturer's suggestedprotocol. Occasionally, 5% DMSO is added to the mixture in repeatexperiments, to facilitate the sequencing of difficult templates.

[0216] Sequencing reactions are performed in a DNA thermal cycler(Perkin Elmer Corporation, Norwalk, Conn.) following the recommendedamplification conditions. Typically, DNA samples were preparedcontaining 500 ng of template DNA and 100 ng of primer of choice inthin-walled 0.2 mL PCR tubes that have been brought to 12 uL withMillipore milli-Q (mQ)-quality water. 2 ul of 2 mM Mg⁺⁺ was added toeach tube. Tubes were denatured for 5 minutes at 96° C. in aPerkin-Elmer System 9700 thermal cycler. After denaturation, the tubeswere chilled to a temperature of 4° C. by the thermal cycler. 6 ul ofABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit wasadded to each tube. The samples were returned to the thermal cycler andcycle-sequenced using the following program: (1) 96° C. for 30 sec; (2)50° C. for 5 sec; (3) 60° C. for 4 min, followed by step (1) for 24additional cycles and then held at 4° C. Cycle sequencing was completeafter about 2.5 hours.

[0217] Samples are purified to remove excess dye terminators with usingCentri-Sep™ spin columns (Princeton Separations, Adelphia, N.J.) orpurified through a Millipore MAHV N45 50 Multiscreen-HV filtration platewhich had been filled with 25 uL Sephadex G-50 superfine resin and 300uL mQ water. Before loading samples onto filtration plates, the platewas prespun in a centrifuge at 750×g for 2 min to remove excess water.The samples were loaded onto the resin and the plate spun again at 750×gfor 4 min. The purified sample was collected into a 96-well plate thatwas placed directly underneath the Sephadex-filled plate during thespin. The liquid in the 96-well plate was dried at room temperature in aSpeed Vac. After 45-60 min the DNA was dried and pelleted at the bottomof the plate. Samples were resuspended in 3 uL of a formamide/blueDextran loading dye and were heated for 2 minutes (see p.33 ofPerkin-Elmer Big Dye manual for loading buffer recipe). Samples wereloaded onto 48 cm well-to-read length 4.5% acrylamide gels and sequencedfor 7 hr using ABI automated DNA sequencers (typically run module SeqRun 48E-1200 and dye set DT, Program BD, Set Any-Primer).

[0218] Overlapping DNA sequence fragments are analyzed and assembledinto master DNA contigs using Sequencher DNA Analysis software (GeneCodes Corporation, Ann Arbor, Mich.) or the Perkin-Elmer Data Collectionand Sequence Analysis programs to assign bases to the data collected.

[0219] BLAST, ClustalW, and Boxshade Homology Alignment Tools

[0220] A variety of programs can be used to align nucleotide or peptidesequences to each other and to facilitate homology searches in largesequence databases. BLAST (Basic Local Alignment Search Tool), whichimplements the statistical matching theory by Karlin and Altschul (Proc.Natl. Acad. Sci. USA 87: 2264-2268, 1990; Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993), is a widely used program for rapidly detectingungapped nucleotide or peptide subsequences that match a given querysequence (Available from the National Center for BiotechnologyInformation, http://www.ncbi.nlm.nih.gov). BLAST uses a heuristicalgorithm which seeks local as opposed to global alignments and istherefore able to detect relationships among sequences which share onlyisolated regions of similarity (Altschul et al., J. Mol. Biol. 215:403-410, 1990).

[0221] Two parameters can be varied which alter the sensitivity andquantity of BLAST search results. Parameter B (with a default value of10) regulates the number of high-scoring segment pairs (alignments)reported in the results. Parameter V (with a default value of 10) is themaximum number of database sequences (hits) for which one-linedescriptions will be reported. Matches are based on high-scoring segmentpairs (HSPs). Two sequences may share more than one HSP, if the HSPs areseparated by gaps. The BLAST algorithm is sensitive to ambiguities inthe sequence and is not well-suited for sequences that contain manygaps.

[0222] The program blastp compares an amino acid query sequence againsta protein sequence database. blastn compares a nucleotide query sequenceagainst a nucleotide sequence database. blastx compares a nucleotidequery sequence translated in all reading frames against a proteinsequence database. You could use this option to find potentialtranslation products of an unknown nucleotide sequence. tblastn comparesa protein query sequence against a nucleotide sequence databasedynamically translated in all reading frames. tblastx compares thesix-frame translations of a nucleotide query sequence against thesix-frame translations of a nucleotide sequence database (Seehttp://www.ncbi.nlm.nih.gov/Education/BLASTinfo/ for more information onBLAST, related programs, and pattern matching algorithms).

[0223] Nucleotides searches performed with BLAST, score=98-557, wordlength 514 letters, were used to obtain nucleotide sequences homologousto nucleic acid molecules of the present invention. Protein searches areperformed with BLASTP, score=50, word length=3 to obtain amino acidsequences homologous to a reference polypeptide (e.g., SEQ ID NO: 2).

[0224] Clustal W version 1.74, which implements a different algorithmfor alignment of multiple DNA or protein sequences, was also used toprepare alignments and to assign percent identities between differentsequences. This program improves the sensitivity of progressive multiplesequence alignment through sequence weighting, position specific gappenalties and weight matrix choice (Thompson et al., Nucleic AcidsResearch, 22(22):4673-4680, 1994). The default parameters for version1.74 were used facilitate alignments and to assign percent identitiesbetween two sequences. The input consisted of sequences in FASTA formatand the output is the alignment shown in the figures. For nucleic acidsequences, the iub DNA weight matrix was used. For amino acid sequences,the blosum protein weight matrix was used (Seehttp://www.ncbi.nlm.nih.gov/Education/BLASTinfo/ for more information onBLAST, related programs, and pattern matching algorithms.

[0225] Boxshade v 3.31 is a public domain program for creating nicelyformatted printouts from muliple-aligned protein or DNA sequences.Boxshade, by itself, does not create alignments, but applies shading orcoloring to files that were previously prepared by other sequencealignment programs. The inputs to Boxshade are the alignments created byClustalW and the threashold values for the residues to be colored orshaded. In most cases, except where specified, a 50% identity value wasused. With this setting, if a position has greater than or equal to halfof the sequences sharing an identical residue, then it is shaded.Boxshade is available by ftp from ftp. or by e-mail from Kay Hofmann(khofmann@isrec-sun1-unil.ch or Michael D. Baron(michael.baron@bbsrc.ac.uk).

[0226] Protein Purification and Characterization

[0227] Protein purification can be accomplished using any of a varietyof chromatographic methods such as: ion exchange, gel filtration orhydrophobic chromatography or reversed phase HPLC. In some cases,proteins which are properly folded can be affinity-purified usingaffinity reagents, such as monoclonal antibodies or receptor subunitsattached to a suitable matrix. These and other protein purificationmethods are described in detail in Methods in Enzymology, Volume 182“Guide to Protein Purification” edited by Murray Deutscher, AcademicPress, San Diego, Calif., 1990.

[0228] The purified protein can be analyzed by RP-HPLC, electrospraymass spectrometry, and SDS-PAGE. Protein quantitation is done by aminoacid composition, RP-HPLC, and/or Bradford protein dye-binding assays.In some cases, tryptic peptide mapping is performed in conjunction withelectrospray mass spectrometry to confirm the identity of the protein.

EXAMPLES

[0229] The following examples will illustrate the invention in greaterdetail, although it will be understood that the invention is not limitedto these specific examples. Various other examples will be apparent tothe person skilled in the art after reading the present disclosurewithout departing from the spirit and scope of the invention. It isintended that all such other examples be included within the scope ofthe appended claims.

Example 1 Preparation of A. ochraceus Spores for RNA Extraction

[0230]Aspergillus ochraceus ATCC 18500 stock culture (50 ul) was grownfor 3-4 days on plates containing sporulation medium: 50 g(L molasses, 5g/L cornsteep liquid, 5 g/L KH₂PO₄, 25 g/L NaCl, 25 g/L glucose, 20 g/Lagar, and 0.4 g/L progesterone, pH 5.8. Progesterone was included in themedia to induce the steroid 11 α-hydroxylase. Spores were scraped fromthe plates into 5 to 7 ml saline, washed in saline, collected bycentrifugation, and suspended in saline containing 15% glycerol. Thespores were frozen on dry ice and stored at −80° C. Approximately 0.8 gspores were incubated at 30° C. in a 1 liter flask containing 400 ml 1%glucose, 50 mM KH₂PO₄ and 0.1 g canrenone, pH 7.0. This treatment priorto spore disruption has three benefits: (1) to induce the steroid 11α-hydroxylase by incubation with canrenone; (2) to determine whether thespores were catalyzing the 11 α-hydroxylation of canrenone; (3) and tosoften the spore wall. After approximately 26 hours of incubating withshaking at 30° C. to provide better aeration, the spores were collectedby centrifugation. Visual inspection with the aid of a microscopeindicated that very few had started to germinate. The spore pellets wereflash frozen in liquid nitrogen and stored at −80° C. The media wasanalyzed for presence of 11 alpha hydroxy canrenone by HPLC to determinewhether spores used for library construction demonstrated the desiredactivity.

Example 2 A. ochraceus Spores Catalyze 11 α-Hydroxylation of Canrenone

[0231] Approximately 160 ml of media from the spore induction wasextracted three times with 70 ml ethyl acetate to collect the steroidsubstrate and products. The organic phase was dried over anhydrousmagnesium sulfate, filtered, and evaporated to dryness. The residue wasdissolved in 8 ml methanol so that the final concentration of canrenonewas approximately 15 mM (assuming quantitative recovery). The mediaextract was diluted 10- to 15-fold into 50% methanol for HPLC analysis.Stock solutions of canrenone and 11 α-hydroxy canrenone were prepared inmethanol. Standards for HPLC analysis were prepared from these stocksolutions by diluting to a final concentration of 750 uM with 50%methanol. Media extract and standards were chromatographed over a C-4reverse phase HPLC column. The media exhibited a component with the sameretention time as the 11 α-hydroxy canrenone standard, as monitored at254 nm (data not shown).

Example 3 Growth of A. ochraceus Mycelia for RNA Extraction

[0232] Liquid cultures of Aspergillus ochraceus mycelia were grown in 10g/L peptone, 10 g/L yeast extract and 10 g/L glucose containing 20 g/Lcanrenone for 24 to 72 hours at 28° C. in a volume of 160 ml. Ten mlsamples of cells were filtered, washed with cold water, frozen, andstored at −80° C.

Example 4 Extraction of Total RNA from Induced Spores

[0233] Approximately 0.4 g spores were disrupted in 40 ml Trizol reagent(Life Technologies, Rockville, Md.) using a Mini-Beadbeater™ model 3110(Biospec Products, Bartlesville, Okla.). Briefly, spore-Trizol mixturewas subjected to four 30 second pulses at low speed. Between pulses,tubes containing spores were chilled on ice. Visual inspection with theaid of a microscope indicated that the majority of the spores weredisrupted by this treatment. The debris was pelleted by low-speedcentrifugation and the total RNA in the supernatant was extractedfollowing the manufacturer's recommended protocols for use with Trizol.Briefly, 2 ml chloroform was added for each 10 ml Trizol in 11 mlpolypropylene centrifuge tubes. Following a 3 minute extraction ofproteins, phase separation was done by centrifugation and the aqueousphase containing the RNA was transferred to a clean tube forprecipitation with an equal volume of isopropanol. The precipitated RNAwas recovered by centrifugation and washed with 70% ethanol. The RNA wasresuspended in 10 ml water, re-extracted with chloroform andprecipitated with ethanol overnight at −20° C. Total RNA (3 mg) wasrecovered by centrifugation and rehydrated in 2 ml water, andprecipitated on ice by adding an equal volume of cold 4 M lithiumchloride. This precipitation was done to remove DNA, carbohydrates,heme, and other impurities which can carry over from guanidine methods.The RNA was recovered by a 25 minute centrifugation.

Example 5 Extraction of Total RNA from Induced Mycelia

[0234] Approximately 0.5 g wet weight cells were pulverized to a finepowder under liquid nitrogen with a mortar and pestle pre-chilled in dryice. The powder was added to 10 ml Trizol Reagent (Life Technologies)and homogenized with a Kinematica polytron (Kinematica AG, Lucerne,Switzerland) at setting #4. Cellular debris was removed bycentrifugation prior to chloroform extraction. The aqueous phasecontaining nucleic acids was precipitated with isopropanol for 10minutes at room temperature. The precipitate was collected bycentrifugation and washed with 70% ethanol. The RNA was rehydrated inwater and re-extracted with chloroform to remove any residual proteins.The aqueous phase was precipitated at −20° C. with 1/10 volume of 3 Msodium acetate and 2.5 volumes absolute ethanol. The final yield was 424ug. Approximately 4 ug and 16 ug of total RNA were separated byelectrophoresis through a 1.2% agarose gel and visualized by staining inethidium bromide. Chromosomal DNA was present as a minor contaminant.

Example 6 Extraction of Total RNA from HepG2 Cells

[0235] Hepatocellular human liver carcinoma cells (HepG2), ATCC HB-8065,were maintained in DMEM high glucose media supplemented with Penstrep,glutamate and 10% fetal bovine serum (Life Technologies, Rockville,Md.). Cells were induced overnight with 0.05% ethanol and harvested forRNA extraction by trypsinization. Briefly, the cell pellet wasresuspended in >10× volumes of 4 M guanidine isothiocyanate, 50 mMTris-HCl, pH 7.5, 25 mM EDTA (solution D, Life Technologies) and thenvortexed. Water and sodium acetate, pH 4.1, were added such that thefinal concentration of sodium acetate was 0.1 M. The RNA solution wasextracted with one half volume of chloroform and placed on ice for 15minutes. The aqueous phase was re-extracted with chloroform andprecipitated overnight with isopropanol. Total RNA was resuspended insolution D and re-precipitated with isopropanol, followed by twoprecipitations in water containing 0.3 M sodium acetate pH 5.5 and 2.5volumes of ethanol. PolyA⁺ selection was performed twice as describedbelow.

Example 7 PolyA⁺ Selection of mRNA

[0236] PolyA⁺ RNA was selected from total RNA with an Eppendorf 5Prime,Inc. kit (Boulder Colo.). Briefly, each 1 mg of total RNA was selectedtwice over a column containing oligo dT cellulose. The column slurry waspacked by gentle centrifugation and equilbrated with 0.5 M NaCl. RNA wasallowed to bind to the dT cellulose for 15 minutes at room temperature.The columns were washed once with 0.5 M NaCl, and twice with 0.1 M NaCl.PolyA⁺ RNA was eluted in 0.5 ml 10 mM Tris-HCl, 1 mM EDTA, pH 7.5. Theselection by oligo dT cellulose was performed twice. The mRNA wasprecipitated at −20° C. with 0.3 M sodium acetate in 50% ethanol, withglycogen added as carrier.

Example 8 cDNA Synthesis and Library Construction

[0237] The Superscript™ Plasmid System for cDNA Synthesis and PlasmidCloning kit (Life Technologies) was used for cDNA systhesis and libraryconstruction. Superscript II reverse transcriptase catalyzed the firststrand of cDNA in a 20 ul reaction for 1 hour at 42° C. The finalcomposition was 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl₂, 10 mMDTT, 50 uM each dATP, dCTP, dGTP and dTTP, 50 ug/ml oligo-dT-NotIprimer-adaptors that were phosphorylated at their 5′ end (LifeTechnologies) and 50,000 units/ml Superscript II reverse transcriptase.oligo-dT-NotI primer-adapter (SEQ ID NO:58)5′-pGACTAGT TCTAGA TCGCGA GCGGCCGC CC (T)₁₅-3′      SpeI    XbaI  NruI    NotI

[0238] A radiolabeled tracer ([α-³²P]dCTP) was not added. The secondstrand of cDNA was synthesized in a reaction volume of 150 ul. The finalcomposition of this mixture including the first strand reaction was 25mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM (NH₄)₂SO₄, 0.15 mM B-NAD⁺, 250 uMeach dATP, dCTP, dGTP and dTTP, 1.2 mM DTT, 65 units/ml E. coli DNAligase, 250 units/ml E. coli DNA polymerase 1 and 13 units/ml E. coliRnase H. After a 2 hour incubation at 16° C., 10 units of T4 DNApolymerase was added, and incubated 5 minutes at 16° C. The reaction wasstopped with 10 ul 0.5 M EDTA and the cDNA was separated from cDNAssmaller than 300 base pairs, primer-adaptors and deoxynucleotides withGENECLEAN II (BIO 101 Inc. La Jolla, Calif.). Annealed Sal I adaptors(Life Technologies) that were phosphorylated at their 5′ blunt end wereligated to the cDNA overnight at 16° C. SalI adapter5′-TCGACCCACGCGTCCG-3′ (SEQ ID NO:59) 3′-GGGTGCGCAGGCp-5′ (SEQ ID NO:60)

[0239] GENECLEAN II was used to remove the adaptors. The cDNA was thendigested with NotI. QIAquick columns (QIAGEN, Valencia, Calif.) wereused to remove small DNA fragments from the cDNA, which was ethanolprecipitated.

Example 9 Size Fractionation of cDNA

[0240] The cDNA was enriched for species approximately 1.5 kb and largerby gel electrophoresis through 0.8% Sea-Plaque agarose (FMC BioProducts,Rockland Me.) in TAE buffer. The preparative gel had a lane of DNA sizemarkers which was excised from the gel after electrophoresis and stainedwith ethidum bromide for visualization under ultraviolet light next to aruler so that the appropriate region of the cDNA could be recovered fromthe gel. GENECLEAN II was used to extract the cDNA, which was eluted in20 ul water.

Example 10 Library Construction in Vector pSport1 and Electroporationinto E. coli

[0241] An aliquot of the size-selected cDNA was ligated overnight at 4°C. with pSport1 (Life Technologies, Inc., Rockville, Md.) predigestedwith NotI and SalI in a 20 ul reaction containing 50 mM Tris-HCl, pH7.6, 10 mM MgCl, 1 mM ATP, 5% (w/v) PEG 8000, 1 mM DTT, 2.5 ug/mlpSport1, approximately 0.5 ug/ml cDNA, and 50 units/ml T4 DNA ligase.The ligation mixture was precipitated by the addition of 12.5 ul 7.5 Mammonium acetate, 5 ul yeast tRNA carrier and 70 ul absolute ethanol.The ligated cDNA was recovered by centrifugation at room temperature for20 minutes and rehydrated in 5 ul sterile water. One ul of the ligatedcDNA was introduced into ElectroMAX DH10B E. coli (Life Technologies) byelectroporation. Cells were allowed to recover in 1 ml SOC medium (LifeTechnologies) for 1 hour at 37° C., before plating an aliquot on LB with100 ug/ml ampicillin. The titer of the Aspergillus ochraceus sporelibrary (designated LIB3025) was determined by preparing serialdilutions of the cell suspension in SOC. The equivalent of 1 ul, 0.1 uland 0.01 ul samples of the cell suspension were plated, and theresulting titer was calculated to be 1.75×10⁶/ml colony forming units.

Example 11 Identification of Clones Encoding Cytochrome P450 Enzymes byDNA Eequence Analysis and Construction of Plasmid pMON45624 EncodingAspergillus ochraceus 11 Alpha Hydroxylase

[0242] Cloning of 11 Alpha Hydroxylase from Aspergillus ochraceus

[0243] Approximately 2,000 colonies were selected on LB agar platescontaining 100 ug/ml ampicillin and miniprep plasmid DNA samples wereprepared for sequencing. Unidirectional sequencing was performed fromthe 3′ end of the expressed sequence tags (ESTs) beginning at the NotIsite encompassing part of the poly dT primer used for cDNA synthesis.Two universal primers were used to facilitate the sequencing: M13reverse: CAG GAA ACA GCT ATG AC (SEQ ID NO:40) T7 promoter: TAA TAC GACTCA CTA TAG GG (SEQ ID NO:41)

[0244] Most known cytochrome p450s contain a conserved heme-bindingregion approximately 50 amino acid residues (150 nucleotides) upstreamof the stop codon (Nelson et al, Pharmacogenetics 6: 1-42, 1996). The2,000 ESTs were screened for sequences encoding the canonicalheme-binding motif (FXXGXXXCXG, where “X” is any amino acid) in theappropriate region using BLASTX and visual inspection of the sequencesscored as hydroxylases for the canonical heme-binding motif. Onlyfifteen ESTs had the heme-binding motif. One EST was unique and theother fourteen appeared to be overlapping sequences. The cDNA insertsfrom seven clones encoding putative cytochrome p450 enzymes were thensequenced to completion. All seven encoded the same enzyme.

[0245] Gene Amplification of Aspergillus ochraceus 11 Alpha Hydroxylase

[0246] The coding region of the 11 alpha hydroxylase was amplified byPCR using a unique clone from the A. ochraceus cDNA spore library(LIB3025) as a template. The primers included recognition sites forEcoRI (forward) and XbaI (reverse) for directional cloning intopFastbac1. Amplification was carried out for 32 cycles using a PCR corekit (Roche) and 50 pmol of each primer. One cycle consisted of adenaturation step at 94° C. for 45 seconds, an annealing step at 60° C.for 45 seconds, and an elongation step at 72° C. for 60 seconds. Primer11alphaOH-for: gatcgaattcATGCCCTTCTTCACTGGGCT (SEQ ID NO:42) Primer11alphaOH-rev: gatctctagaTTACACAGTTAAACTCGCCATATCGAT (SEQ ID NO:43)

[0247] Construction of pMON45624

[0248] The amplified fragments described above were purified through aQIAquick column (Qiagen, Valencia Calif.) and digested with EcoRI andXbaI prior to ligation into pFastBac1 cleaved with EcoRI and XbaI. Theresulting plasmid was designated pMON45624 and the DNA sequence verifiedusing primers based on the vector sequence and internal primers based onthe 11 alpha hydroxylase sequence (shown below). Primer Bacfwd:CTGTTTTCGTAACAGTTTTG (SEQ ID NO:44) Primer PolyA: CCTCTACAAATGTGGTATG(SEQ ID NO:45) Primer GAGATCAAGATTGCCTT (SEQ ID NO:46) 45624-for1:Primer CTTCGACGCTCTCAA (SEQ ID NO:47) 45624-for2: PrimerGCAATCTTGATCTCGTT (SEQ ID NO:48) 45624-rev1:

[0249] The nucleotide and predicted amino acid sequences of the cloned11 alpha hydroxylase are displayed in FIG. 1 as SEQ ID NO: 1 and SEQ IDNO: 2, respectively.

[0250]FIG. 4 sets forth an amino acid homology alignment of A. ochraceus11 alpha hydroxylase cloned in pMON45624 and aligned with relatedenzymes found in GenBank using BLAST. FIG. 5 is a phylogenetic treeshowing the this relationship graphically. FIG. 6 shows the percenthomology between Aspergillus ochraceus steroid 11 alpha hydroxylase andthe top 10 enzymes found in GenBank using BLAST, calculated usingClustal W and Boxshade.

Example 12 Amplification of cDNA Encoding Human NADPH Cytochrome P450Reductase and Cloning Into Plasmids pMON45603, pMON45604, and pMON45605

[0251] Gene Amplification of Human Oxidoreductase

[0252] Approximately 1 ug polyA⁺ mRNA from HepG2 cells was heated to 65°C. for 10 minutes with 100 ng random hexamers (Invitrogen, Carlsbad,Calif.) in an 11 ul reaction. The mixture was chilled on ice, thenincubated at 42° C. for 75 minutes in a 20 ul reaction containing 1 ulRNase inhibitor (Promega, Madison, Wis.), 0.01 M DTT, 5 mM dNTPs, 50 mMTris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl₂ and 1 ul SuperScriptII enzyme(Life Technologies). The reverse transcriptase was inactivated byheating to 95° C. for 2 minutes. First strand cDNA was stored at −20° C.Forward and reverse primers were based on the nucleotide sequence ofaccession number S90469 (human placental partial mRNA encodingcytochrome P450 reductase (SEQ ID NO: 49)). The accession number of thecorresponding protein sequence is AAB21814 (SEQ ID NO: 50). The humanoxidoreductase was cloned in two pieces which were assembled inpFastBac1 (Life Technologies) by ligation at an internal HincII site.The primers included restriction sites for directional subcloning intopFastBac1. Primer H. oxred 1A: gatcggatccaatATGGGAGACTCCCACGTGGACAC (SEQID NO:07) Primer H. oxred 1B: CAGCTGGTTGACGAGAGCAGAG (SEQ ID NO:08)Primer H. oxred 2A: CTCTGCTCTCGTCAACCAGCTG (SEQ ID NO:09) Primer H.oxred 2B: gatcggtaccttaGCTCCACACGTCCAGGGAGTAG (SEQ ID NO:10)

[0253] The second strand was synthesized using 400 uM dNTP and 167 nM ofeach primer set per 150 ul reaction. Amplification was performed withDeep Vent polymerase (New England Biolabs, Beverly, Mass.). The reactionfor segment 2 (the 3′ half of the oxidoreductase cDNA) was adjusted to5% DMSO. The amplification included an initial cycle of denaturation at94° C. for 90 seconds, followed by annealing at 62° C. for 2 minutes andelongation at 72° C. for 2 minutes. This was followed by 30 cycles,consisting of a 45 second denaturation step, a 45 second annealing step,and a 60 second elongation step. The elongation step was extended to 5minutes for the final cycle.

[0254] Construction ofpMON45603, pMON45604, pMON45605

[0255] The PCR fragments for the 5′ half of the oxidoreductase cDNA weredigested with BamHI and HincII. The PCR fragments for the 3′ half of theoxidoreductase cDNA were digested with HincII and KpnI and ligated intopBluescript II (Stratagene, La Jolla, Calif.) for sequencing. Theresulting plasmids were designated pMON45603 (5′ segment) and pMON45604(3′ segment). The BamHI/HincII fragment from pMON45603 and theHincII/KpnI fragment from pMON45604 were ligated into pFastbac1 cut withBamHI and KpnI, to generate pMON45605.

[0256] Sequencing primers were based on the sequence of GenBankaccession number S90469 (SEQ ID NO 49), a cDNA encoding cytochrome P450reductase [human, placenta, mRNA Partial, 2403 nt]. The cognate proteinsequence is: AAB21814 (SEQ ID NO 50) cytochrome P450 reductase {EC1.6.2.4} [human, placenta, Peptide Partial, 676 aa] [Homo sapiens]. ThecDNA insert of pMON45603 was sequenced using primer oxred 1C, and thecDNA insert of pMON45604 was sequenced using primer oxred 2C and 2D.Universal T7 (SEQ ID NO: 41) and M13 reverse (SEQ ID NO: 40) primers,which annealed to vector sequences flanking the cDNA inserts were alsoused for sequencing. Primer oxred 1C: GTGGACCACAAGCTCGTACTG (SEQ IDNO:61) Primer oxred 2C: CATCGACCACCTGTGTGAGCTG (SEQ ID NO:62) Primeroxred 2D: GTACAGGTAGTCCTCATCCGAG (SEQ ID NO:63)

[0257] The nucleotide and predicted amino acid sequences of the clonedhuman oxidoreductase are displayed in FIG. 2 as SEQ ID NO: 3 and SEQ IDNO: 4, respectively. FIG. 11 sets forth an alignment of humanoxidoreductase with top 4 hits from SwissProt. FIG. 12 sets forth aphylogenetic tree displaying the genetic relatedness of humanoxidoreductase, to these hits. FIG. 13 shows the percent identitybetween human oxidoreductase and top 4 hits from SwissProt.

Example 13 Amplification of cDNA Encoding NADPH Cytochrome P450Reductase from A ochraceus and Cloning into Plasmids pMON45630,pMON45631, and pMON45632.

[0258] Gene Amplification of Aspergillus ochraceus Oxidoreductase

[0259] An alignment of sequences from the Aspergillus niger cprA geneaccession number Z26938 (SEQ ID NO: 65) and a partial cDNA clone804561639F1 from Aspergillus fumigatus (PathoSeq Database, IncytePharmaceuticals) was visually scanned to select regions of high homologyfor the design of primers for PCR. A primer set was selected whichspanned the coding region of the cprA gene product from amino acids 203to 693.

[0260] Primers were selected from the 5′ most region of overlap wherethe amino acid sequence was identical between both and the nucleic acidsequence differed by 2 positions in the 3rd codon position. For the 3′primer, the nucleic acid encoding the stop codon, last 7 amino acidresidues and 2 additional bases corresponding to second and thirdpositions in the codon of the amino acid residue 8 positions from thestop codon encodes ARG in A. niger and SER in A. fumigatis (CGC vs.AGC). Inosines replaced the third base in codons when there was adiscrepancy between the A. niger and A. fumigatus sequence. PrimerA.oxred-for1: GACGG I GC I GGTACAATGGA (SEQ ID NO:11) PrimerA.oxred-rev1: TTA I GACCA I ACATC I TCCTGGTAGC (SEQ ID NO:12) (where I=Inosine)

[0261] A partial cDNA clone was amplified from approximately 5 ug oftotal RNA extracted from A. ochraceus mycelia. Before the first strandsynthesis, the RNA was heated to 65° C. for 10 minutes with 100 ngrandom hexamers (Promega Madison Wis.) in an 11 ul reaction mixture. Themixture was chilled on ice, then incubated at 42° C. for 75 minutes in a20 ul reaction containing 1 ul RNase inhibitor (Promega), 0.01 M DTT, 5mM dNTPs, 50 mM Tris-HCl, pH 8.3), 75 mM KCl, 3 mM MgCl₂ and 1 ulSuperScriptII (LTI). The reverse transcriptase was inactivated byheating to 95° C. for 2 minutes. The first strand cDNA was stored at−20° C. The second strand was synthesized using 5 ul of the first strandas template. The reaction included 500 nM primers, 200 uM each dNTP, andTaq polymerase and buffer as supplied in PCR core kit (Roche MolecularBiochemicals, Indianapolis, Ind.). Amplification was performed using 32cycles of a 30 second denaturation step at 94° C., a 30 second annealingstep at 60° C. and a 60 second elongation step at 72° C. The amplifiedDNA products were cloned into pGEM-T (Promega, Madison, Wis.) andsequenced using universal T7 (SEQ ID NO: 41) and SP6 (SEQ ID NO: 57)primers.

[0262] Primer SP6 GATTTAGGTGACACTATAG (SEQ ID NO: 57)

[0263] Alignment of the sequences with the A. niger cprA gene revealedthat the A. ochraceus clones had an intron in the same position as theintron in the A. niger gene. This indicated that the A. ochraceus PCRproducts might have been amplified from a genomic DNA contaminant of thetotal RNA. A reverse primer based on the A. ochraceus sequence wasdesigned to amplify the approximately 600 missing bp including theinitial methionine. The A. ochraceus cDNA library was then used as atemplate for PCR. The forward primer was based on the reverse complementof vector pSport1 (Life Technologies) bases 299 to 326. The otherprimer, A.oxred-rev2 was bases on the A. ochraceus sequence encodingresidues 326-333. Primer pSport-for1: CAAGCTCTAATACGACTCACTATAGGGA (SEQID NO: 13) Primer A.oxred-rev2: CAGGAACCGATCGACCTCGGAA (SEQ ID NO: 14)

[0264] The A. ochraceus spore library size made from gel-purifiedfragments >1.5 kb in size was then used as a template for amplifying thefinal 200 bases of coding region. Two new reverse primers were designedfrom the A.oxred sequence, and a new forward primer based on pSport1(bases 295-328) was also used. Primer A.oxred-rev3:GTCACCCTCACCAGCAGAGCCAATG (SEQ ID NO: 15) Primer A.oxred-rev4:CCACATTGCGAACCATAGCGTTGTAGTG (SEQ ID NO: 16) Primer pSport-for2:GCCAAGCTCTAATACGACTCACTATAGGGAAAGC (SEQ ID NO: 17)

[0265] Amplification was performed using an Elongase polymerase kit(Life Technologies, Rockville Md.) for 35 cycles consisting of adenaturation step at 94° C. for 30 seconds, an annealing step at 63° C.for 30 seconds, and an elongation step at 68° C. for 5 minutes. The PCRproducts were cloned directly into pCR11 TOPO (Invitrogen). Twelveclones were sequenced, and the composite sequence, extended for 232bases upstream of the initial methionine, and included 2 in-frame stopcodons (Data not shown).

[0266] Primers incorporating the complete coding region of A.oxred weredesigned with a 5′ SalI site and a 3′ XhoI site for ligation intoexpression vector pFastBac1. Primer A.oxred-for2:gtcgacATGGCGCAACTCGATACTCTC (SEQ ID NO: 18) Primer A.oxred-rev5:ctcgagttaGGACCAGACATCGTCCTGGTAG (SEQ ID NO: 19)

[0267]A. ochraceus total RNA was used as a template for PCR with theseprimers and the Elongase kit. Amplification consisted of 35 cycles witha 30 second denaturation step at 94° C., a 30 second annealing step at64° C., and a 5 minute elongation step at 68° C. An aliquot of the cDNAfrom reaction ran as a single band of approximately 2.1 kb.

[0268] Construction of pMON45630

[0269] The PCR products were cloned directly into pCR11-TOPO(Invitrogen, Carlsbad, Calif.). All clones contained the internal intronnoted earlier. One clone was designated pMON45630.

[0270] Construction ofpMON45631 and pMON45632

[0271] A strategy based on two step PCR from an internal BamHI siteapproximately 170 bp upstream of the 5′ splice site was employed togenerate clones lacking the intron. Primer A.oxred-for3:GGATCCCTCGCGACCTGTGATCAT (SEQ ID NO: 20) Primer A.oxred-for4:CGAAGATTTCTTGTACAAGGATGAATGGAAGACTTTTC (SEQ ID NO: 21) PrimerA.oxred-rev6: CTGAAAAGTCTTCCATTCATCCTTGTACAAGAAATC (SEQ ID NO: 22)

[0272] Primers A.oxred-for4 and rev6 were complementary and flanked theintron. The first PCR reaction used an A.oxred clone linearized at theinternal BamHI site as template. Polymerase and buffers were supplied bythe PCR core kit (Roche Molecular Biochemicals, Indianapolis, Ind.).Primer and dNTP concentrations were 500 nM and 200 uM, respectively. Tworeactions were performed, using a combination of A.oxred-for3 withA.oxred-rev6, and A.oxred-for4 with A.oxred-rev5. Following a 2 minuteinitial denaturation, 28 cycles of PCR amplification were performed. Onecycle included a 45 second denaturation at 94° C., a 45 seconddenaturation step at 62° C. and a 45 second elongation step at 72° C.One ul of each reaction served as template for the second PCRamplification with primers A.oxred-for3 and A.oxred-rev5 using Elongaseenzyme and buffers. Amplification consisted of 30 cycles with a 30second denaturation step at 94° C., a 30 second annealing step at 62°C., and a 5 minute elongation step at 68° C. The PCR products weredirectly cloned into pCR11-TOPO. DNA sequencing demonstrated that theintron had been removed. This clone was designated pMON45631.

[0273] Plasmid pMON45632 was constructed in a three-way ligation byligating the SalI/BamHI fragment from pMON45630 with the BamHI/XhoIfragment from pMON45631 and vector pFastBac1, which had been cut withSalI and XhoI and de-phosphorylated to enhance the recovery of vectorswith the desired inserts.

[0274] The nucleotide and amino acid sequences of the cloned Aspergillusochraceus 11 oxidoreductase are displayed in FIG. 3 as SEQ ID NO: 5 andSEQ ID NO: 6, respectively. FIG. 7 sets forth the amino acid homology ofAspergillus ochraceus and human oxidoreductase to NADPH cytochrome P450reductases from A. niger, mouse, and S. cerevisiae. FIG. 8 sets forththe amino acid alignment for A. ochraceus, A. niger , and S. cerevisiaeoxidoreductases. FIG. 9 is a phylogenetic tree showing the relatednessof Aspergillus ochraceus and human oxidoreductase to reductases from A.niger, yeast, and mouse. FIG. 10 shows the percent homology betweenAspergillus ochraceus steroid 11 alpha hydroxylase and theoxidoreductases from A. niger, yeast, and mouse, calculated usingClustal W and Boxshade.

Example 15 Generation of Polyclonal Antibodies Recognizing Aspergillusochraceus 11 Alpha Hydroxylase and Aspergillus ochraceus NADPHCytochrome p450 Reductase

[0275] Generation of Anti-11-α-Hydroxylase Antibodies

[0276] Polyclonal antibodies against Aspergillus ochraceus 11 alphahydroxylase and NADPH cytochrome p450 reductase were raised in rabbitsagainst synthetic peptides (prepared by Sigma/Genosis, The Woodlands,TX) corresponding to several regions of the following predicted proteinsequences: 11aOH peptide 1: AAAYWLATLQPSDLPELN (SEQ ID NO: 23) 11aOHpeptide 2: CRQILTPYIHKRKSLKGTTD (SEQ ID NO: 24) 11aOH peptide 3:HMGFGHGVHACPGRFFASNEI (SEQ ID NO: 25) oxr peptide 1:CTYWAVAKDPYASAGPAMNG (SEQ ID NO: 26)

[0277] The 11aOH peptide 2 (SEQ ID NO: 24) corresponds to the G helix,G/H loop, and H helix region present in an alignment of the amino acidsequence of 11 alpha hydroxylase with the corresponding sequence ofCYP3A4 described by Wang and Lu, (Drug Metab. Dispos. 25(6), 762-767,1997). The 11aOH peptide 3 (SEQ ID NO: 25) corresponded to the peptidefragment from the heme-binding domain.

[0278] Immunological grade peptides were monitored for purity usingreverse phase high performance liquid chromatography (HPLC). Eachpeptide was conjugated to keyhole limpet hemacyanin (KLH) and suspendedin Complete Freund's Adjuvant. The conjugated peptide was then injectedsubcutaneously at multiple sites into rabbits. Each conjugated peptidewas injected into two rabbits. All subsequent immunizations were givenin incomplete Freund's Adjuvant. In general, five subsequent injectionswere given at two-week intervals following the initial immunization. IgGfractions were affinity-purified using a Sepharose-Protein A column.Fractions from the two rabbits injected with each peptide were combinedat a 1:1 ratio. The pooled anti-11 alpha hydroxylase (rabbits GN1187/1188) was 0.34 mg/ml IgG. The pooled anti-oxred (rabbits GN2023/2024) was 0.26 mg/ml IgG. The combined IgGs were each diluted 1:10,1:100 and 1:1,000 for a pilot experiment to determine which was dilutionwas optimal for probing Western blots. The 1:10 dilution gave the bestresults and was used for probing subsequent Westerns.

Example 16 Insect Cell Infection and Heterologous Expression

[0279] Proteins were expressed in Sf9 insect cells using baculovirusshuttle vectors (Luckow et al., J. Virol. 67: 4566-4579, 1993). Thebaculovirus shuttle vector (bacmid) contains a mini-F replicon forexpression in bacterial cells, a kanamycin resistance marker forselection, and attTn7 (the target site for the bacterial Tn7 transposon)within the lacZa sequence. Each of these elements is inserted into thepolyhedrin locus of the Autographa californica nuclear polyhedrosisvirus (AcNPV, the native baculovirus) genome. A donor plasmid(pFastBac1, Life Technologies) was used to deliver the gene to beexpressed and was inserted into the bacmid via the bacterial Tn7transposition elements. pFastBac1 contains the Tn7 left and right endsflanking the polyhedrin promoter, a polylinker cloning sequence, theSV40 polyA transcription termination sequence, and the gentamicinresistance gene for selection. Recombinant viruses were generatedfollowing transformation of the pFastBac1 plasmid, which contained asingle 11 alpha hydroxylase or oxidoreductase cDNA, into DH10Bac E. colicells (Life Technologies) that contained the bacmid and helper plasmid.

[0280] Transfections were performed using CellFectin™ reagent (LifeTechnologies) following the manufacturer's protocol for Spodopterafrugigperda (Sf9) cells. Cells were seeded in 6-well tissue cultureplates at 9×10⁵ cells per well in SF-900 serum-free medium (LifeTechnologies) and allowed to attach for at least one hour. Thetransfection mixtures were made following the addition of 5 ul miniprepDNA and 5 μl Cellfectin to polystyrene tubes that contained 200 ulSF-900 medum. The mixtures were allowed to incubate for 15-30 minutes atroom temperature. Prior to transfection, 800 ul SF-900 medium was addedto each tube. The cells were washed one time with 2 ml SF-900 medium,and the DNA mixtures were added to the cells. The cultures were allowedto incubate for 5 hours at 27° C. Following the 5 hr incubation period,the transfection mixture was removed and the cultures were replenishedwith 3 ml per well IPL-41 medium (Life Technologies) supplemented with10% fetal bovine serum. Following a three day incubation period, thecells were harvested, centrifuged, and the supernatant that containedrecombinant virus (designated as passage 1 or P1 stock) was removed andstored at 4° C. A larger viral stock was made by infecting 100 ml freshSf9 cells at 5×10⁵ cells per ml with 0.5 ml of the P1 medium. Thislarger (P2) stock was then titered using a plaque assay protocol(O'Reilly et al., 1992), and used for production of the 11 alphahydroxylase or oxidoreductase enzymes, separately or in combination witheach other.

[0281]FIG. 14 sets forth an immunoblot illustrating expression ofAspergillus ochraceus P450 11 alpha hydroxylase in baculovirus-infectedinsect cells harvested at 25 and 48 hours post infection. Thenitrocellulose membrane was probed with a 1:1 mixture of antibodiesprepared two rabbits immunized with a conjugated synthetic peptide 11aOHpeptide 2 (SEQ ID NO 24).

[0282]FIG. 15 sets forth an immunoblot illustrating expression ofAspergillus ochraceus P450 oxidoreductase in baculovirus-infected insectcells harvested at 25 and 48 hours post infection. The nitrocellulosemembrane was probed with a 1:1 mixture of antibodies prepared tworabbits immunized with a conjugated synthetic peptide oxr peptide 1 (SEQID NO 26).

Example 17 Co-Infection Baculoviruses Expressing of Aspergillusochraceus 11 Alpha Hydroxylase and Human Oxidoreductase

[0283] Sf9 cells were co-infected with virus particles that containedthe steroid 11 alpha hydroxylase cDNA and a separate virus containing ahuman NADPH P450-oxidoreductase. Both viruses were added at amultiplicity of infection (MOI) ratio of 0.1:0.01 (11 aOH to oxr). Oneday after infection, 0.9 μg/ml hemin chloride was added to the culture.The cells were harvested by centrifugation three days after infection(unless specified differently), and the washed cell pellets were frozenuntil processed for sub-cellular fractions.

Example 18 Co-Infection Baculoviruses Expressing of Aspergillusochraeeus 11 Alpha Hydroxylase and Aspergillus ochraceus Oxidoreductase

[0284] Sf9 cells are co-infected with virus particles that contain thesteroid 11 alpha hydroxylase cDNA and a separate virus containing A.ochraceus NADPH P450-oxidoreductase. Both viruses are added at amultiplicity of infection (MOI) ratio of 0.1:0.01 (11 aOH to oxr). Oneday after infection, 0.9 μg/ml hemin chloride is added to the culture.The cells are harvested by centrifugation three days after infection(unless specified differently), and the washed cell pellets are frozenuntil needed in subsequent experiments that require processing into forsub-cellular fractions.

Example 19 Preparation of Subcellular Fractions fromBaculovirus-Infected Insect Cells

[0285] One half gram of the cell pastes from infected sf9 cells anduninfected control cells were thawed and suspended in 40 ml of 0.25 Msucrose with 10 mM KHPO4, adjusted to pH 7.4. The suspensions werehomogenized using a Fisher Sonic Dismembrator, model 300 probe sonicator(Fisher Scientific, St. Louis, Mo.). The samples were transferred to aconical centrifuge tube (Corning Costar Corporation, Cambridge, Mass.)and subjected to centrifugation at 500×g at 5° C. for 15 minutes. Thepellets were resuspended in the same volume of fresh buffer and viewedunder a microscope to confirm complete lysis. Few or no whole cells wereobserved. The supernatants were then subjected to centrifugation at10,000×g for 30 minutes at 5° C. to collect mitochondria, Golgi andother subcellular organelles. The pellets were resuspended in freshbuffer and subjected to centrifugation at 7,800×g for 30 minutes at 5°C. to collect mitochondria.

[0286] The mitochondrial pellets were resuspended in buffer as describedabout and the centrifugation was repeated. The mitochondrial pelletswere resuspended in 2 ml buffered sucrose solution and stored at −80° C.in 100 ul aliquots.

[0287] The supernatants from the original mitochondrial fractionationwere subjected to centrifugation at 200,000×g for 1 hour at 5° C. Themicrosomal pellets were resuspended in 2 ml buffered sucrose solutionand stored at −80° C. in 100 ul aliquots.

[0288] Microsomal Incubations

[0289] Incubation mixtures consisted of Sf9 microsomes (1.0 mg ofprotein/mL final concentration), an NADPH-generating system and 250 uMsubstrate (AD) in 100 mM potassium phosphate buffer, pH 7.4 or 150 mMHEPES buffer, pH 7.4. The NADPH-generating system was composed of thefollowing at the indicated final concentrations: MgCl₂ (7.5 mM),D-glucose-6-phosphate (7.5 mM), NADP (0.80 mM), and glucose-6-phosphatedehydrogenase (1.0 units/mL). Incubations were carried out for theindicated times at 37° C. in a water bath. Following incubation,reactions were terminated by the addition of 0.3 ml methanol. Thesamples were vortexed three times for two seconds and placed on ice, orstored at −70° C. for later analysis.

Example 20 HPLC Assays to Measure Conversion of Steroid Substrates toTheir Hydroxylated Counterparts

[0290] High Performance Liquid Chromatography (HPLC)

[0291] The HPLC method used to separate hydroxylated steroid compoundsfrom steroid substrates, such as 11α-hydroxyandrostenedione fromandrostenedione, is a modified version of the testosterone hydroxylaseassay, described by Sonderfan et al., Arch. Biochem. Biophys. 255:27-41, 1987). The standards for androstenedione and11-beta-hydroxyandrostenedione were obtained from Sigma.11-alpha-hydroxyandrostenedione (89.5% pure, with the major impuritybeing androstenedione) was provided by Searle Medicinal Chemistry. HPLCgrade water and methanol were obtained from Burdick & Jackson.

[0292] The HPLC system consisted of a Model 1050 series pump,autoinjector and variable wavelength detector (Hewlett-Packard,Naperville, Ill.), and a Model TC-50 temperature controller and ModelCH-30 column heater (both Eppendorf, Madison, Wis.).

[0293] Cell membrane fractions derived from insect cells transfectedwith recombinant baculoviruses expressing 11-hydroxylase andcomplementary electron transport proteins were analyzed for11-hydroxylase activity in a reaction mixture containing 80 mM phosphatebuffer, pH 7.4, 8 mM MgCl₂, and 0.9 mM NADP⁺ in a final volume of 200ul. In order to insure an adequate source of reducing equivalents, anNADPH regenerating system was provided by adding glucose-6-phosphatedehydrogenase (1.5 U/ml) and 8 mM glucose-6-phosphate. Steroid substrate(e.g., androstenedione) was provided at a final concentration of 0.3 mM.Reaction mixtures were incubated at 37° C. for 30 min. The reactionswere terminated by the addition of 200 ul methanol and then placed onice. Samples were pelleted by centrifugation to remove precipitatedprotein.

[0294] On one occasion, the incubation was carried out in a volume of0.5 ml in siliconized polypropylene 1.5 ml microcentrifuge tubes at 37°C. for 120 minutes. The enzyme, prepared from microsomal ormitochondrial fractions, was added and the substrate added at aconcentration of 250 μM (e.g., 25 mM methanol stock solution of AD). Thecofactor buffer was 100 mM potassium phosphate, pH 7.4, 7.5 mM MgCl₂,7.5 mM glucose-6-phosphate, 0.80 mM NADP, and 1.0 units/mLglucose-6-phosphate dehydrogenase. HPLC samples were prepared byterminating the 0.5 ml reaction mixture by addition of 0.3 ml methanol,vortexing three times for 2 seconds and storing on ice. The tubes werespun for 5 minutes at −20,000×g in a microcentrifuge and the samplestransferred to autosampler vials and capped.

[0295] Steroid components present in reaction mixtures and media extractwere separated and analyzed by reverse phase HPLC using a 250 mm×4 mmVydac analytical C-4 column. Chromatograms were developed using asolvent gradient from 40% to 100% methanol over a ten minute time periodand holding at 100% methanol for 5 minutes before re-equilibration toinitial conditions. The column effluent was monitored for UV absorbanceat both 254 and 220 nm.

[0296] Androstenedione, testosterone and monohydroxylatedandrostenedione metabolites were resolved on a Nova-pak C18 column, 4micron, 3.9×150 mm (Waters Chromatography, Milford, Mass.) equipped witha 0.22 micron Rheodyne precolumn filter at 40° C. and 1.0 ml mobilephase/min. A stepped gradient was utilized with water as mobile phasesolvent A and methanol as solvent B. The initial concentration ofsolvent B was 42% for 6 min. The percentage of B was increased linearlyto 45% over 4 minutes and then held for 3 minutes. The percentage of Bwas then increased linearly to 80% over 10 minutes and held there for anadditional 2 minutes for a total run time of 25 minutes. The ultravioletdetection wavelength was 247 nm and the injection volume was 200 ul.

[0297] Both the “mitochondria” sample and the “microsomal” sampleproduced peaks matching the HPLC retention time of the11α-hydroxyandrostenedione standard, while other fractions did not.These “mitochondria” and “microsomal” peaks were 3.2 and 2.3%,respectively, of the total peak area quantitated at 247 nm. The11α-hydroxyandrostenedione standard was also spiked into a blankmicrosomal incubation sample at a concentration of 5.0 μg/mL. Theconcentration of the “mitochondria” and “microsomal”11α-hydroxyandrostenedione peaks were 1.75 and 1.31 μg/mL, aftercorrecting for the purity of the standard (89.5%). These concentrationsrepresent 2.3 and 1.7% of substrate converted to11α-hydroxyandrostenedione, using a substrate concentration of 250 μM.

[0298]FIG. 16 sets forth an HPLC tracing illustrating the conversion ofandrostenedione (AD) to its 11 alpha hydroxy counterpart afterincubating AD with subcellular fractions prepared frombaculovirus-infected insect cells expressing Aspergillus ochraceus 11alpha hydroxylase and human oxidoreductase.

Example 21 Recognition of Aspergillus ochraceus 11 Alpha Hydroxylase andAspergillus ochraceus NADPH Cytochrome p450 Reductase by ImmunoblottingUsing Polyclonal Antibodies Generated Against Synthetic Peptides

[0299] Proteins from Sf9 cell lysates (obtained from uninfected andrecombinant baculovirus-infected cells) were loaded onto lanes of a 10%gradient acrylamide mini gel (BioRad, Hercules, Calif.) at equalconcentrations (10 μg per well). The proteins were separated byelectrophoresis at 16 mAmps constant current for approximatley 1 hr in aTris-glycine buffer containing 0.1% SDS (Sigma, St. Louis, Mo.). Theproteins were transferred to nitrocellulose (Schleicher & Schuell,Keene, NH) for 40 min at 70 mAmp constant current. Primary antibodieswere diluted 1:10 (from stock concentrations of 0.34 mg/ml IgG foranti-11 alpha hydroxylase (antibodies GN-1187 and GN-1188 prepared frompeptide 11aOH peptide 2 CRQILTPYIHKRKSLKGTTD (SEQ ID NO: 24)), and 0.26mg/ml IgG for anti-oxred (antibodies GN-2023 and GN-12024 prepared fromoxr peptide 1 CTYWAVAKDPYASAGPAMNG (SEQ ID NO: 26)) and used to probethe nitrocellulose membrane. The antigens were detected usinganti-rabbit horseradish peroxidase (HRP)-linked secondary antibody asrecommended by the manufacturer (New England Biolabs, Beverly, Mass.).Chemilumiescence was detected using luminol and peroxide reagents (NewEngland Biolabs, Beverly, Mass.) following the protocol provided by thevendor. Light emission was recorded using X-OMAT AR film (Eastman KodakCompany, Rochester, N.Y.). Images were recorded using a Minolta Dimage Vdigital camera (Minolta Corporation, Ramsey, N.J.).

Example 22 Characterization of the Aspergillus ochraceus Genomic DNAEncoding 11 Alpha Hydroxylase and Oxidoreductase

[0300] The approaches described above can be used to facilitate theidentification of genes encoding steroid hydroxylases andoxidoreductases within the genome of Aspergillus ochraceus and closelyrelated microorganisms, including Aspergillus niger and Aspergillusnidulans. Other preferred organisms are Rhizopus oryzae, Rhizopusstolonifer, Streptomyces fradiae, Bacillus megaterium, Pseudomonascruciviae, Trichothecium roseum, Fusarium oxysporum f sp. cepae,Rhizopus arrhizus, and Monosporium olivaceum. Other preferred organismsthat are known to have steroid 11 alpha hydroxylase activity aredescribed in the detailed description of the invention, above.

[0301] Briefly, genomic DNA is prepared and shotgun cloned into low copyartificial chromosomes propagated in bacteria. A large number of clonesare sequenced to ensure statistical representation of the entire genome,and the sequences of overlapping clones merged to produce the final mapand sequence of the genome. Analysis of the open reading frames, willreveal regions which are homologous to the steroid hydroxylase andoxidoreductase genes of the present invention, and regions of thetranslated open reading frames which are homologous to these enzymesusing programs designed to facilitate multiple sequence alignments ofnucleotide and protein sequence data such as BLAST, CLUSTAL W, andBoxShade. Genes which encode these proteins are obtained from theartificial chromosomes and recloned into expression vectors such aspFastBac1, transformed into appropriate host cells, which are assayedfor the presence of enzymes capable of carrying out the conversion ofsteroid substrates to their oxidized counterparts.

[0302] It is intended that the scope of the present invention bedetermined by reference to the appended claims. It is recognized that anumber of variations can be made to this invention as it is currentlydescribed but which do not depart from the scope and spirit of theinvention without compromising any of its advantages. These includeisolation of homologous genes from microorganisms known to carry out 11alpha hydroxylation of steroid substrates, preferably fungi andbacteria. This invention is also directed to any substitution ofanalogous components. This includes, but is not restricted to use ofthese techniques to isolate other P450s which are involved insteroidogenesis, including hydroxylases that act at other positions inthe core molecule, and use of these enzymes to facilitate bioconversionof steroid intermediates in modified host microorganisms.

[0303] All references, patents, or applications cited herein areincorporated by reference in their entirety, as if written herein.

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1 65 1 1776 DNA Aspergillus ochraceus CDS (146)...(1690) Aspergillusochraceus 11 alpha hydroxylase 1 tggaagtttt tacacttatt atgccggagccgaaagattc tgagtcgagg ggttggggaa 60 caacactata agacctacaa ccacttggatttggtgaatt tacacgggca ttatcaaaac 120 agccacaagc tgacagctca ttatc atg cccttc ttc act ggg ctt ctg gcg 172 Met Pro Phe Phe Thr Gly Leu Leu Ala 1 5att tac cat agt ctc ata ctc gac aac cca gtc caa acc ctg agc acc 220 IleTyr His Ser Leu Ile Leu Asp Asn Pro Val Gln Thr Leu Ser Thr 10 15 20 25att gtc gta ttg gcg gca gcg tac tgg ctc gca acg ctc cag ccg agc 268 IleVal Val Leu Ala Ala Ala Tyr Trp Leu Ala Thr Leu Gln Pro Ser 30 35 40 gacctt cct gag ctg aat ccc gcc aaa cca ttc gag ttc acc aat cgt 316 Asp LeuPro Glu Leu Asn Pro Ala Lys Pro Phe Glu Phe Thr Asn Arg 45 50 55 cgt cgtgtt cat gag ttt gtt gaa aat agt aag agc ttg ctt gct cgg 364 Arg Arg ValHis Glu Phe Val Glu Asn Ser Lys Ser Leu Leu Ala Arg 60 65 70 ggg agg gaattg cac ggg cac gag ccg tac aga ctc atg tct gaa tgg 412 Gly Arg Glu LeuHis Gly His Glu Pro Tyr Arg Leu Met Ser Glu Trp 75 80 85 gga tcc ttg attgtc ctg ccc cca gag tgc gcc gac gag ctg cgc aac 460 Gly Ser Leu Ile ValLeu Pro Pro Glu Cys Ala Asp Glu Leu Arg Asn 90 95 100 105 gac cca agaatg gac ttt gag acg ccc acc acc gac gac tcc cac gga 508 Asp Pro Arg MetAsp Phe Glu Thr Pro Thr Thr Asp Asp Ser His Gly 110 115 120 tat atc cctggc ttc gac gct ctc aac gca gac ccg aac ctg act aaa 556 Tyr Ile Pro GlyPhe Asp Ala Leu Asn Ala Asp Pro Asn Leu Thr Lys 125 130 135 gtg gtc accaag tac ctc aca aaa gca ttg aac aag ctt act gct ccg 604 Val Val Thr LysTyr Leu Thr Lys Ala Leu Asn Lys Leu Thr Ala Pro 140 145 150 atc tcg catgaa gcg tcc atc gcc atg aaa gcg gtg ctg ggt gac gat 652 Ile Ser His GluAla Ser Ile Ala Met Lys Ala Val Leu Gly Asp Asp 155 160 165 cca gat tggcgt gag atc tac cca gcc aga gac ttg ctc cag ctc gtc 700 Pro Asp Trp ArgGlu Ile Tyr Pro Ala Arg Asp Leu Leu Gln Leu Val 170 175 180 185 gcc cggatg tcg aca aga gtg ttc ctt ggc gag gaa atg tgc aat aac 748 Ala Arg MetSer Thr Arg Val Phe Leu Gly Glu Glu Met Cys Asn Asn 190 195 200 cag gattgg atc caa acc tca tca caa tac gcg gcc ctt gcc ttc ggt 796 Gln Asp TrpIle Gln Thr Ser Ser Gln Tyr Ala Ala Leu Ala Phe Gly 205 210 215 gtc ggtgac aag ctt aga ata tac ccg aga atg atc aga ccg ata gta 844 Val Gly AspLys Leu Arg Ile Tyr Pro Arg Met Ile Arg Pro Ile Val 220 225 230 cat tggttc atg cca tcc tgt tgg gag ctg cgc cga tcg ctg cga cgc 892 His Trp PheMet Pro Ser Cys Trp Glu Leu Arg Arg Ser Leu Arg Arg 235 240 245 tgc cgacag att ctc acg ccg tac att cac aaa cgc aag tcc ctg aag 940 Cys Arg GlnIle Leu Thr Pro Tyr Ile His Lys Arg Lys Ser Leu Lys 250 255 260 265 gggacc acg gac gag cag ggc aag ccc ctt atg ttt gat gat tcc atc 988 Gly ThrThr Asp Glu Gln Gly Lys Pro Leu Met Phe Asp Asp Ser Ile 270 275 280 gagtgg ttc gag cga gag ctg ggt ccc aac cac gac gcg gtc ctg aag 1036 Glu TrpPhe Glu Arg Glu Leu Gly Pro Asn His Asp Ala Val Leu Lys 285 290 295 caggtc acg ctc tcc ata gtt gct atc cac acc acg agt gac cta ctc 1084 Gln ValThr Leu Ser Ile Val Ala Ile His Thr Thr Ser Asp Leu Leu 300 305 310 ttgcag gcc atg agc gat ctc gcg cag aac ccg aaa gtg cta caa gca 1132 Leu GlnAla Met Ser Asp Leu Ala Gln Asn Pro Lys Val Leu Gln Ala 315 320 325 gtgcgc gag gag gtg gtc cga gtg ctg agc acc gag ggg ctc agc aag 1180 Val ArgGlu Glu Val Val Arg Val Leu Ser Thr Glu Gly Leu Ser Lys 330 335 340 345gtc tcg ctt cac agt ctc aag ctc atg gac agc gcg ttg aag gaa agc 1228 ValSer Leu His Ser Leu Lys Leu Met Asp Ser Ala Leu Lys Glu Ser 350 355 360cag cgt ctc agg cct acg ctt ctc ggc tcc ttt cgt cgg cag gca acg 1276 GlnArg Leu Arg Pro Thr Leu Leu Gly Ser Phe Arg Arg Gln Ala Thr 365 370 375aat gac atc aag ctg aag agc ggg ttt gtc ata aag aaa ggg act aga 1324 AsnAsp Ile Lys Leu Lys Ser Gly Phe Val Ile Lys Lys Gly Thr Arg 380 385 390gtc gtg atc gac agc acc cat atg tgg aat ccc gag tat tac act gac 1372 ValVal Ile Asp Ser Thr His Met Trp Asn Pro Glu Tyr Tyr Thr Asp 395 400 405cct ctc cag tac gac ggg tac cgc tac ttc aac aag cgg cag aca ccc 1420 ProLeu Gln Tyr Asp Gly Tyr Arg Tyr Phe Asn Lys Arg Gln Thr Pro 410 415 420425 ggc gag gac aag aac gcg ttg ctc gtc agc aca agc gcc aac cac atg 1468Gly Glu Asp Lys Asn Ala Leu Leu Val Ser Thr Ser Ala Asn His Met 430 435440 gga ttc ggt cac ggc gtt cac gcc tgt cct ggc aga ttc ttc gcc tcc 1516Gly Phe Gly His Gly Val His Ala Cys Pro Gly Arg Phe Phe Ala Ser 445 450455 aac gag atc aag att gcc ttg tgt cat atc atc tta aat tat gag tgg 1564Asn Glu Ile Lys Ile Ala Leu Cys His Ile Ile Leu Asn Tyr Glu Trp 460 465470 cgt ctt cca gac ggc ttc aag ccc cag cct ctc aac atc ggg atg act 1612Arg Leu Pro Asp Gly Phe Lys Pro Gln Pro Leu Asn Ile Gly Met Thr 475 480485 tat ctg gcg gat ccc aat acc agg atg ctg atc agg cca cgc aag gcg 1660Tyr Leu Ala Asp Pro Asn Thr Arg Met Leu Ile Arg Pro Arg Lys Ala 490 495500 505 gag atc gat atg gcg agt tta act gtg tag gtcgaacacg aagtcctgat1710 Glu Ile Asp Met Ala Ser Leu Thr Val * 510 gaagtgttat tggtcagtgggtgaagcaag tcgcagaaat gtgtaacaat ttataagaat 1770 aaaaaa 1776 2 514 PRTAspergillus ochraceus 2 Met Pro Phe Phe Thr Gly Leu Leu Ala Ile Tyr HisSer Leu Ile Leu 1 5 10 15 Asp Asn Pro Val Gln Thr Leu Ser Thr Ile ValVal Leu Ala Ala Ala 20 25 30 Tyr Trp Leu Ala Thr Leu Gln Pro Ser Asp LeuPro Glu Leu Asn Pro 35 40 45 Ala Lys Pro Phe Glu Phe Thr Asn Arg Arg ArgVal His Glu Phe Val 50 55 60 Glu Asn Ser Lys Ser Leu Leu Ala Arg Gly ArgGlu Leu His Gly His 65 70 75 80 Glu Pro Tyr Arg Leu Met Ser Glu Trp GlySer Leu Ile Val Leu Pro 85 90 95 Pro Glu Cys Ala Asp Glu Leu Arg Asn AspPro Arg Met Asp Phe Glu 100 105 110 Thr Pro Thr Thr Asp Asp Ser His GlyTyr Ile Pro Gly Phe Asp Ala 115 120 125 Leu Asn Ala Asp Pro Asn Leu ThrLys Val Val Thr Lys Tyr Leu Thr 130 135 140 Lys Ala Leu Asn Lys Leu ThrAla Pro Ile Ser His Glu Ala Ser Ile 145 150 155 160 Ala Met Lys Ala ValLeu Gly Asp Asp Pro Asp Trp Arg Glu Ile Tyr 165 170 175 Pro Ala Arg AspLeu Leu Gln Leu Val Ala Arg Met Ser Thr Arg Val 180 185 190 Phe Leu GlyGlu Glu Met Cys Asn Asn Gln Asp Trp Ile Gln Thr Ser 195 200 205 Ser GlnTyr Ala Ala Leu Ala Phe Gly Val Gly Asp Lys Leu Arg Ile 210 215 220 TyrPro Arg Met Ile Arg Pro Ile Val His Trp Phe Met Pro Ser Cys 225 230 235240 Trp Glu Leu Arg Arg Ser Leu Arg Arg Cys Arg Gln Ile Leu Thr Pro 245250 255 Tyr Ile His Lys Arg Lys Ser Leu Lys Gly Thr Thr Asp Glu Gln Gly260 265 270 Lys Pro Leu Met Phe Asp Asp Ser Ile Glu Trp Phe Glu Arg GluLeu 275 280 285 Gly Pro Asn His Asp Ala Val Leu Lys Gln Val Thr Leu SerIle Val 290 295 300 Ala Ile His Thr Thr Ser Asp Leu Leu Leu Gln Ala MetSer Asp Leu 305 310 315 320 Ala Gln Asn Pro Lys Val Leu Gln Ala Val ArgGlu Glu Val Val Arg 325 330 335 Val Leu Ser Thr Glu Gly Leu Ser Lys ValSer Leu His Ser Leu Lys 340 345 350 Leu Met Asp Ser Ala Leu Lys Glu SerGln Arg Leu Arg Pro Thr Leu 355 360 365 Leu Gly Ser Phe Arg Arg Gln AlaThr Asn Asp Ile Lys Leu Lys Ser 370 375 380 Gly Phe Val Ile Lys Lys GlyThr Arg Val Val Ile Asp Ser Thr His 385 390 395 400 Met Trp Asn Pro GluTyr Tyr Thr Asp Pro Leu Gln Tyr Asp Gly Tyr 405 410 415 Arg Tyr Phe AsnLys Arg Gln Thr Pro Gly Glu Asp Lys Asn Ala Leu 420 425 430 Leu Val SerThr Ser Ala Asn His Met Gly Phe Gly His Gly Val His 435 440 445 Ala CysPro Gly Arg Phe Phe Ala Ser Asn Glu Ile Lys Ile Ala Leu 450 455 460 CysHis Ile Ile Leu Asn Tyr Glu Trp Arg Leu Pro Asp Gly Phe Lys 465 470 475480 Pro Gln Pro Leu Asn Ile Gly Met Thr Tyr Leu Ala Asp Pro Asn Thr 485490 495 Arg Met Leu Ile Arg Pro Arg Lys Ala Glu Ile Asp Met Ala Ser Leu500 505 510 Thr Val 3 2031 DNA human CDS (1)...(2031) humanoxidoreductase 3 atg gga gac tcc cac gtg gac acc agc tcc acc gtg tcc gaggcg gtg 48 Met Gly Asp Ser His Val Asp Thr Ser Ser Thr Val Ser Glu AlaVal 1 5 10 15 gcc gaa gaa gta tct ctt ttc agc atg acg gac atg att ctgttt tcg 96 Ala Glu Glu Val Ser Leu Phe Ser Met Thr Asp Met Ile Leu PheSer 20 25 30 ctc atc gtg ggt ctc cta acc tac tgg ttc ctc ttc aga aag aaaaaa 144 Leu Ile Val Gly Leu Leu Thr Tyr Trp Phe Leu Phe Arg Lys Lys Lys35 40 45 gaa gaa gtc ccc gag ttc acc aaa att cag aca ttg acc tcc tct gtc192 Glu Glu Val Pro Glu Phe Thr Lys Ile Gln Thr Leu Thr Ser Ser Val 5055 60 aga gag agc agc ttt gtg gaa aag atg aag aaa acg ggg agg aac atc240 Arg Glu Ser Ser Phe Val Glu Lys Met Lys Lys Thr Gly Arg Asn Ile 6570 75 80 atc gtg ttc tac ggc tcc cag acg ggg act gca gag gag ttt gcc aac288 Ile Val Phe Tyr Gly Ser Gln Thr Gly Thr Ala Glu Glu Phe Ala Asn 8590 95 cgc ctg tcc aag gac gcc cac cgc tac ggg atg cga ggc atg tca gcg336 Arg Leu Ser Lys Asp Ala His Arg Tyr Gly Met Arg Gly Met Ser Ala 100105 110 gac cct gag gag tat gac ctg gcc gac ctg agc agc ctg cca gag atc384 Asp Pro Glu Glu Tyr Asp Leu Ala Asp Leu Ser Ser Leu Pro Glu Ile 115120 125 gac aac gcc ctg gtg gtt ttc tgc atg gcc acc tac ggt gag gga gac432 Asp Asn Ala Leu Val Val Phe Cys Met Ala Thr Tyr Gly Glu Gly Asp 130135 140 ccc acc gac aat gcc cag gac ttc tac gac tgg ctg cag gag aca gac480 Pro Thr Asp Asn Ala Gln Asp Phe Tyr Asp Trp Leu Gln Glu Thr Asp 145150 155 160 gtg gat ctc tct ggg gtc aag ttc gcg gtg ttt ggt ctt ggg aacaag 528 Val Asp Leu Ser Gly Val Lys Phe Ala Val Phe Gly Leu Gly Asn Lys165 170 175 acc tac gag cac ttc aat gcc atg ggc aag tac gtg gac aag cggctg 576 Thr Tyr Glu His Phe Asn Ala Met Gly Lys Tyr Val Asp Lys Arg Leu180 185 190 gag cag ctc ggc gcc cag cgc atc ttt gag ctg ggg ttg ggc gacgac 624 Glu Gln Leu Gly Ala Gln Arg Ile Phe Glu Leu Gly Leu Gly Asp Asp195 200 205 gat ggg aac ttg gag gag gac ttc atc acc tgg cga gag cag ttctgg 672 Asp Gly Asn Leu Glu Glu Asp Phe Ile Thr Trp Arg Glu Gln Phe Trp210 215 220 ccg gcc gtg tgt gaa cac ttt ggg gtg gaa gcc act ggc gag gagtcc 720 Pro Ala Val Cys Glu His Phe Gly Val Glu Ala Thr Gly Glu Glu Ser225 230 235 240 agc att cgc cag tac gag ctt gtg gtc cac acc gac ata gatgcg gcc 768 Ser Ile Arg Gln Tyr Glu Leu Val Val His Thr Asp Ile Asp AlaAla 245 250 255 aag gtg tac atg ggg gag atg ggc cgg ctg aag agc tac gagaac cag 816 Lys Val Tyr Met Gly Glu Met Gly Arg Leu Lys Ser Tyr Glu AsnGln 260 265 270 aag ccc ccc ttt gat gcc aag aat ccg ttc ctg gct gca gtcacc acc 864 Lys Pro Pro Phe Asp Ala Lys Asn Pro Phe Leu Ala Ala Val ThrThr 275 280 285 aac cgg aag ctg aac cag gga acc gag cgc cac ctc atg cacctg gaa 912 Asn Arg Lys Leu Asn Gln Gly Thr Glu Arg His Leu Met His LeuGlu 290 295 300 ttg gac atc tcg gac tcc aaa atc agg tat gaa tct ggg gaccac gtg 960 Leu Asp Ile Ser Asp Ser Lys Ile Arg Tyr Glu Ser Gly Asp HisVal 305 310 315 320 gct gtg tac cca gcc aac gac tct gct ctc gtc aac cagctg ggc aaa 1008 Ala Val Tyr Pro Ala Asn Asp Ser Ala Leu Val Asn Gln LeuGly Lys 325 330 335 atc ctg ggt gcc gac ctg gac gtc gtc atg tcc ctg aacaac ctg gat 1056 Ile Leu Gly Ala Asp Leu Asp Val Val Met Ser Leu Asn AsnLeu Asp 340 345 350 gag gag tcc aac aag aag cac cca ttc ccg tgc cct acgtcc tac cgc 1104 Glu Glu Ser Asn Lys Lys His Pro Phe Pro Cys Pro Thr SerTyr Arg 355 360 365 acg gcc ctc acc tac tac ctg gac atc acc aac ccg ccgcgt acc aac 1152 Thr Ala Leu Thr Tyr Tyr Leu Asp Ile Thr Asn Pro Pro ArgThr Asn 370 375 380 gtg ctg tac gag ctg gcg cag tac gcc tcg gag ccc tcggag cag gag 1200 Val Leu Tyr Glu Leu Ala Gln Tyr Ala Ser Glu Pro Ser GluGln Glu 385 390 395 400 ctg ctg cgc aag atg gcc tcc tcc tcc ggc gag ggcaag gag ctg tac 1248 Leu Leu Arg Lys Met Ala Ser Ser Ser Gly Glu Gly LysGlu Leu Tyr 405 410 415 ctg agc tgg gtg gtg gag gcc cgg agg cac atc ctggcc atc ctg cag 1296 Leu Ser Trp Val Val Glu Ala Arg Arg His Ile Leu AlaIle Leu Gln 420 425 430 gac tgc ccg tcc ctg cgg ccc ccc atc gac cac ctgtgt gag ctg ctg 1344 Asp Cys Pro Ser Leu Arg Pro Pro Ile Asp His Leu CysGlu Leu Leu 435 440 445 ccg cgc ctg cag gcc cgc tac tac tcc atc gcc tcatcc tcc aag gtc 1392 Pro Arg Leu Gln Ala Arg Tyr Tyr Ser Ile Ala Ser SerSer Lys Val 450 455 460 cac ccc aac tct gtg cac atc tgt gcg gtg gtt gtggag tac gag acc 1440 His Pro Asn Ser Val His Ile Cys Ala Val Val Val GluTyr Glu Thr 465 470 475 480 aag gcc ggc cgc atc aac aag ggc gtg gcc accaac tgg ctg cgg gcc 1488 Lys Ala Gly Arg Ile Asn Lys Gly Val Ala Thr AsnTrp Leu Arg Ala 485 490 495 aag gag cct gcc ggg gag aac ggc ggc cgt gcgctg gtg ccc atg ttc 1536 Lys Glu Pro Ala Gly Glu Asn Gly Gly Arg Ala LeuVal Pro Met Phe 500 505 510 gtg cgc aag tcc cag ttc cgc ctg ccc ttc aaggcc acc acg cct gtc 1584 Val Arg Lys Ser Gln Phe Arg Leu Pro Phe Lys AlaThr Thr Pro Val 515 520 525 atc atg gtg ggc ccc ggc acc ggg gtg gca cccttc ata ggc ttc atc 1632 Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro PheIle Gly Phe Ile 530 535 540 cag gag cgg gcc tgg ctg cga cag cag ggc aaggag gtg ggg gag acg 1680 Gln Glu Arg Ala Trp Leu Arg Gln Gln Gly Lys GluVal Gly Glu Thr 545 550 555 560 ctg ctg tac tac ggc tgc cgc cgc tcg gatgag gac tac ctg tac cgg 1728 Leu Leu Tyr Tyr Gly Cys Arg Arg Ser Asp GluAsp Tyr Leu Tyr Arg 565 570 575 gag gag ctg gcg cag ttc cac agg gac ggtgcg ctc acc cag ctc aac 1776 Glu Glu Leu Ala Gln Phe His Arg Asp Gly AlaLeu Thr Gln Leu Asn 580 585 590 gtg gcc ttc tcc cgg gag cag tcc cac aaggtc tac gtc cag cac ctg 1824 Val Ala Phe Ser Arg Glu Gln Ser His Lys ValTyr Val Gln His Leu 595 600 605 cta aag caa gac cga gag cac ctg tgg aagttg atc gaa ggc ggt gcc 1872 Leu Lys Gln Asp Arg Glu His Leu Trp Lys LeuIle Glu Gly Gly Ala 610 615 620 cac atc tac gtc tgt ggg gat gca cgg aacatg gcc agg gat gtg cag 1920 His Ile Tyr Val Cys Gly Asp Ala Arg Asn MetAla Arg Asp Val Gln 625 630 635 640 aac acc ttc tac gac atc gtg gct gagctc ggg gcc atg gag cac gcg 1968 Asn Thr Phe Tyr Asp Ile Val Ala Glu LeuGly Ala Met Glu His Ala 645 650 655 cag gcg gtg gac tac atc aag aaa ctgatg acc aag ggc cgc tac tcc 2016 Gln Ala Val Asp Tyr Ile Lys Lys Leu MetThr Lys Gly Arg Tyr Ser 660 665 670 ctg gac gtg tgg agc 2031 Leu Asp ValTrp Ser 675 4 677 PRT human 4 Met Gly Asp Ser His Val Asp Thr Ser SerThr Val Ser Glu Ala Val 1 5 10 15 Ala Glu Glu Val Ser Leu Phe Ser MetThr Asp Met Ile Leu Phe Ser 20 25 30 Leu Ile Val Gly Leu Leu Thr Tyr TrpPhe Leu Phe Arg Lys Lys Lys 35 40 45 Glu Glu Val Pro Glu Phe Thr Lys IleGln Thr Leu Thr Ser Ser Val 50 55 60 Arg Glu Ser Ser Phe Val Glu Lys MetLys Lys Thr Gly Arg Asn Ile 65 70 75 80 Ile Val Phe Tyr Gly Ser Gln ThrGly Thr Ala Glu Glu Phe Ala Asn 85 90 95 Arg Leu Ser Lys Asp Ala His ArgTyr Gly Met Arg Gly Met Ser Ala 100 105 110 Asp Pro Glu Glu Tyr Asp LeuAla Asp Leu Ser Ser Leu Pro Glu Ile 115 120 125 Asp Asn Ala Leu Val ValPhe Cys Met Ala Thr Tyr Gly Glu Gly Asp 130 135 140 Pro Thr Asp Asn AlaGln Asp Phe Tyr Asp Trp Leu Gln Glu Thr Asp 145 150 155 160 Val Asp LeuSer Gly Val Lys Phe Ala Val Phe Gly Leu Gly Asn Lys 165 170 175 Thr TyrGlu His Phe Asn Ala Met Gly Lys Tyr Val Asp Lys Arg Leu 180 185 190 GluGln Leu Gly Ala Gln Arg Ile Phe Glu Leu Gly Leu Gly Asp Asp 195 200 205Asp Gly Asn Leu Glu Glu Asp Phe Ile Thr Trp Arg Glu Gln Phe Trp 210 215220 Pro Ala Val Cys Glu His Phe Gly Val Glu Ala Thr Gly Glu Glu Ser 225230 235 240 Ser Ile Arg Gln Tyr Glu Leu Val Val His Thr Asp Ile Asp AlaAla 245 250 255 Lys Val Tyr Met Gly Glu Met Gly Arg Leu Lys Ser Tyr GluAsn Gln 260 265 270 Lys Pro Pro Phe Asp Ala Lys Asn Pro Phe Leu Ala AlaVal Thr Thr 275 280 285 Asn Arg Lys Leu Asn Gln Gly Thr Glu Arg His LeuMet His Leu Glu 290 295 300 Leu Asp Ile Ser Asp Ser Lys Ile Arg Tyr GluSer Gly Asp His Val 305 310 315 320 Ala Val Tyr Pro Ala Asn Asp Ser AlaLeu Val Asn Gln Leu Gly Lys 325 330 335 Ile Leu Gly Ala Asp Leu Asp ValVal Met Ser Leu Asn Asn Leu Asp 340 345 350 Glu Glu Ser Asn Lys Lys HisPro Phe Pro Cys Pro Thr Ser Tyr Arg 355 360 365 Thr Ala Leu Thr Tyr TyrLeu Asp Ile Thr Asn Pro Pro Arg Thr Asn 370 375 380 Val Leu Tyr Glu LeuAla Gln Tyr Ala Ser Glu Pro Ser Glu Gln Glu 385 390 395 400 Leu Leu ArgLys Met Ala Ser Ser Ser Gly Glu Gly Lys Glu Leu Tyr 405 410 415 Leu SerTrp Val Val Glu Ala Arg Arg His Ile Leu Ala Ile Leu Gln 420 425 430 AspCys Pro Ser Leu Arg Pro Pro Ile Asp His Leu Cys Glu Leu Leu 435 440 445Pro Arg Leu Gln Ala Arg Tyr Tyr Ser Ile Ala Ser Ser Ser Lys Val 450 455460 His Pro Asn Ser Val His Ile Cys Ala Val Val Val Glu Tyr Glu Thr 465470 475 480 Lys Ala Gly Arg Ile Asn Lys Gly Val Ala Thr Asn Trp Leu ArgAla 485 490 495 Lys Glu Pro Ala Gly Glu Asn Gly Gly Arg Ala Leu Val ProMet Phe 500 505 510 Val Arg Lys Ser Gln Phe Arg Leu Pro Phe Lys Ala ThrThr Pro Val 515 520 525 Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro PheIle Gly Phe Ile 530 535 540 Gln Glu Arg Ala Trp Leu Arg Gln Gln Gly LysGlu Val Gly Glu Thr 545 550 555 560 Leu Leu Tyr Tyr Gly Cys Arg Arg SerAsp Glu Asp Tyr Leu Tyr Arg 565 570 575 Glu Glu Leu Ala Gln Phe His ArgAsp Gly Ala Leu Thr Gln Leu Asn 580 585 590 Val Ala Phe Ser Arg Glu GlnSer His Lys Val Tyr Val Gln His Leu 595 600 605 Leu Lys Gln Asp Arg GluHis Leu Trp Lys Leu Ile Glu Gly Gly Ala 610 615 620 His Ile Tyr Val CysGly Asp Ala Arg Asn Met Ala Arg Asp Val Gln 625 630 635 640 Asn Thr PheTyr Asp Ile Val Ala Glu Leu Gly Ala Met Glu His Ala 645 650 655 Gln AlaVal Asp Tyr Ile Lys Lys Leu Met Thr Lys Gly Arg Tyr Ser 660 665 670 LeuAsp Val Trp Ser 675 5 2322 DNA Aspergillus ochraceus CDS (233)...(2320)Aspergillus ochraceus oxidoreductase 5 cttatttcgt ttaggaagag caccggcttcggtgtccttc cttaccctct tattcttcct 60 cttctgactc cctttttgtt attgatcgcccatctcggtg aacatttggg atatctttcc 120 ctctccccct cccgccccga ccctccttatcttctcctcc cgtccagcat ttagctcgcc 180 atcgaattcg caattccttc ctcgtgactcttcatcgctg agcgtcctca tc atg gcg 238 Met Ala 1 caa ctc gat act ctc gatttg gtc gtc ctg gtg gcg ctc ttg gtg ggt 286 Gln Leu Asp Thr Leu Asp LeuVal Val Leu Val Ala Leu Leu Val Gly 5 10 15 agc gtg gcc tac ttc acc aagggc acc tac tgg gcc gtc gcc aaa gac 334 Ser Val Ala Tyr Phe Thr Lys GlyThr Tyr Trp Ala Val Ala Lys Asp 20 25 30 cct tat gcc tcg gct ggt ccg gcgatg aat gga ggc gcc aag gcc ggc 382 Pro Tyr Ala Ser Ala Gly Pro Ala MetAsn Gly Gly Ala Lys Ala Gly 35 40 45 50 aag act cgc gac att gtt cag aaaatg gac gaa act ggc aaa aac tgt 430 Lys Thr Arg Asp Ile Val Gln Lys MetAsp Glu Thr Gly Lys Asn Cys 55 60 65 gtg att ttc tac ggc tcg caa acc ggtacc gct gag gac tac gcg tcc 478 Val Ile Phe Tyr Gly Ser Gln Thr Gly ThrAla Glu Asp Tyr Ala Ser 70 75 80 aga ctg gcc aag gaa ggc tcc cag cga ttcggt ctc aag acc atg gtg 526 Arg Leu Ala Lys Glu Gly Ser Gln Arg Phe GlyLeu Lys Thr Met Val 85 90 95 gcc gat ctg gag gac tac gac tac gaa aac ctggaa aag ttc ccc gag 574 Ala Asp Leu Glu Asp Tyr Asp Tyr Glu Asn Leu GluLys Phe Pro Glu 100 105 110 gac aag gtt gtt ttc ttc gtt ctg gcc act tatggc gag ggt gaa ccc 622 Asp Lys Val Val Phe Phe Val Leu Ala Thr Tyr GlyGlu Gly Glu Pro 115 120 125 130 acg gat aat gcg gtt gaa ttc tac cag ttcgtc acg ggc gaa gat gct 670 Thr Asp Asn Ala Val Glu Phe Tyr Gln Phe ValThr Gly Glu Asp Ala 135 140 145 gct ttc gag agc ggc gct acc gcc gac gataag cct ctg tct tct ctc 718 Ala Phe Glu Ser Gly Ala Thr Ala Asp Asp LysPro Leu Ser Ser Leu 150 155 160 aag tat gtc acg ttt ggt ctg ggt aac aacacc tat gag cac tac aac 766 Lys Tyr Val Thr Phe Gly Leu Gly Asn Asn ThrTyr Glu His Tyr Asn 165 170 175 gct atg gtt cgc aat gtg gac gcc gct ctcaca aag ttc ggc gcc caa 814 Ala Met Val Arg Asn Val Asp Ala Ala Leu ThrLys Phe Gly Ala Gln 180 185 190 cgc att ggc tct gct ggt gag ggt gac gacggc gct ggt aca atg gaa 862 Arg Ile Gly Ser Ala Gly Glu Gly Asp Asp GlyAla Gly Thr Met Glu 195 200 205 210 gag gat ttc ctg gcc tgg aag gaa cccatg tgg gct gcc ctt tct gag 910 Glu Asp Phe Leu Ala Trp Lys Glu Pro MetTrp Ala Ala Leu Ser Glu 215 220 225 gcg atg aac ctg caa gag cgc gat gcggtc tac gag ccg gtc ttc aat 958 Ala Met Asn Leu Gln Glu Arg Asp Ala ValTyr Glu Pro Val Phe Asn 230 235 240 gtc acc gag gac gag tcc ctg agc cccgaa gat gag aac gtt tac ctc 1006 Val Thr Glu Asp Glu Ser Leu Ser Pro GluAsp Glu Asn Val Tyr Leu 245 250 255 ggt gag ccc act caa ggt cat ctc caaggc gag ccc aag ggc ccg tac 1054 Gly Glu Pro Thr Gln Gly His Leu Gln GlyGlu Pro Lys Gly Pro Tyr 260 265 270 tct gcg cac aac ccg ttc atc gct cccatc tcc gaa tct cgt gaa ctg 1102 Ser Ala His Asn Pro Phe Ile Ala Pro IleSer Glu Ser Arg Glu Leu 275 280 285 290 ttc aac gtc aag gac cgc aac tgtctg cac atg gaa atc agc atc gcc 1150 Phe Asn Val Lys Asp Arg Asn Cys LeuHis Met Glu Ile Ser Ile Ala 295 300 305 ggt agc aac ctc act tac cag actggt gac cac atc gct gtt tgg ccc 1198 Gly Ser Asn Leu Thr Tyr Gln Thr GlyAsp His Ile Ala Val Trp Pro 310 315 320 acc aac gcc ggt tcc gag gtc gatcgg ttc ctg cag gct ttt ggt ctc 1246 Thr Asn Ala Gly Ser Glu Val Asp ArgPhe Leu Gln Ala Phe Gly Leu 325 330 335 gaa gga aag cgc cac tcc gtc atcaac att aag ggt atc gat gtg acc 1294 Glu Gly Lys Arg His Ser Val Ile AsnIle Lys Gly Ile Asp Val Thr 340 345 350 gct aag gtt ccg att ccc act cctacg acc tat gac gcc gca gtt cgc 1342 Ala Lys Val Pro Ile Pro Thr Pro ThrThr Tyr Asp Ala Ala Val Arg 355 360 365 370 tac tac ctg gaa gtc tgt gccccc gtt tcc cgt cag ttt gtc tcg act 1390 Tyr Tyr Leu Glu Val Cys Ala ProVal Ser Arg Gln Phe Val Ser Thr 375 380 385 ctc gct gcc ttt gcc cct gatgaa gcg acc aag gcg gag atc gtt cgt 1438 Leu Ala Ala Phe Ala Pro Asp GluAla Thr Lys Ala Glu Ile Val Arg 390 395 400 ttg ggt ggc gac aag gac tatttc cat gag aag att acc aac cga tgc 1486 Leu Gly Gly Asp Lys Asp Tyr PheHis Glu Lys Ile Thr Asn Arg Cys 405 410 415 ttc aac atc gct cag gct ctccag agc atc acg tcc aag cct ttc acc 1534 Phe Asn Ile Ala Gln Ala Leu GlnSer Ile Thr Ser Lys Pro Phe Thr 420 425 430 gcc gtc ccg ttc tcc ctg cttatc gaa ggt atc acc aag ctt cag ccc 1582 Ala Val Pro Phe Ser Leu Leu IleGlu Gly Ile Thr Lys Leu Gln Pro 435 440 445 450 cgt tac tac tcg atc tcctcg tct tcc ctg gtt cag aag gac aag att 1630 Arg Tyr Tyr Ser Ile Ser SerSer Ser Leu Val Gln Lys Asp Lys Ile 455 460 465 agc att acc gcc gtt gtggag tcg gtt cgc ttg cct ggt gag gaa cac 1678 Ser Ile Thr Ala Val Val GluSer Val Arg Leu Pro Gly Glu Glu His 470 475 480 att gtc aag ggt gtg accacg aac tat ctt ctc gcg ctc aag gaa aag 1726 Ile Val Lys Gly Val Thr ThrAsn Tyr Leu Leu Ala Leu Lys Glu Lys 485 490 495 caa aac ggc gag cct tcccct gac ccg cac ggc ttg act tac tct atc 1774 Gln Asn Gly Glu Pro Ser ProAsp Pro His Gly Leu Thr Tyr Ser Ile 500 505 510 act gga ccc cgt aac aagtac gat ggc atc cat gtc ccc gtt cac gtc 1822 Thr Gly Pro Arg Asn Lys TyrAsp Gly Ile His Val Pro Val His Val 515 520 525 530 cgc cac tcg aac ttcaaa ttg ccc tcg gat ccc tcg cga cct gtg atc 1870 Arg His Ser Asn Phe LysLeu Pro Ser Asp Pro Ser Arg Pro Val Ile 535 540 545 atg gtt gga ccc ggtact ggt gtt gct cct ttc cgt ggg ttt atc cag 1918 Met Val Gly Pro Gly ThrGly Val Ala Pro Phe Arg Gly Phe Ile Gln 550 555 560 gag cgt gct gcc ttggcc gcg aag ggc gag aag gtc gga act acc ttg 1966 Glu Arg Ala Ala Leu AlaAla Lys Gly Glu Lys Val Gly Thr Thr Leu 565 570 575 ctt ttc ttc ggc tgccgt aag tcc gac gaa gat ttc ttg tac aag gat 2014 Leu Phe Phe Gly Cys ArgLys Ser Asp Glu Asp Phe Leu Tyr Lys Asp 580 585 590 gaa tgg aag act tttcag gag cag ctt ggc gac tcg ctc aag atc atc 2062 Glu Trp Lys Thr Phe GlnGlu Gln Leu Gly Asp Ser Leu Lys Ile Ile 595 600 605 610 act gcc ttc tctcgt gaa tcg gct gag aaa gtc tac gtc cag cac agg 2110 Thr Ala Phe Ser ArgGlu Ser Ala Glu Lys Val Tyr Val Gln His Arg 615 620 625 ctg cgt gag catgcc gag ctg gtc agt gac ctg ctg aag cag aaa gcc 2158 Leu Arg Glu His AlaGlu Leu Val Ser Asp Leu Leu Lys Gln Lys Ala 630 635 640 act ttc tat gtttgc ggt gac gct gcc aac atg gcc cgt gaa gtc aac 2206 Thr Phe Tyr Val CysGly Asp Ala Ala Asn Met Ala Arg Glu Val Asn 645 650 655 ctc gtg ctt gggcaa atc att gcc aag cag cgc ggt ctc cct gcc gag 2254 Leu Val Leu Gly GlnIle Ile Ala Lys Gln Arg Gly Leu Pro Ala Glu 660 665 670 aag ggc gag gagatg gtg aag cac atg cgc agc agc ggc agc tac cag 2302 Lys Gly Glu Glu MetVal Lys His Met Arg Ser Ser Gly Ser Tyr Gln 675 680 685 690 gac gat gtctgg tcc taa aa 2322 Asp Asp Val Trp Ser * 695 6 695 PRT Aspergillusochraceus 6 Met Ala Gln Leu Asp Thr Leu Asp Leu Val Val Leu Val Ala LeuLeu 1 5 10 15 Val Gly Ser Val Ala Tyr Phe Thr Lys Gly Thr Tyr Trp AlaVal Ala 20 25 30 Lys Asp Pro Tyr Ala Ser Ala Gly Pro Ala Met Asn Gly GlyAla Lys 35 40 45 Ala Gly Lys Thr Arg Asp Ile Val Gln Lys Met Asp Glu ThrGly Lys 50 55 60 Asn Cys Val Ile Phe Tyr Gly Ser Gln Thr Gly Thr Ala GluAsp Tyr 65 70 75 80 Ala Ser Arg Leu Ala Lys Glu Gly Ser Gln Arg Phe GlyLeu Lys Thr 85 90 95 Met Val Ala Asp Leu Glu Asp Tyr Asp Tyr Glu Asn LeuGlu Lys Phe 100 105 110 Pro Glu Asp Lys Val Val Phe Phe Val Leu Ala ThrTyr Gly Glu Gly 115 120 125 Glu Pro Thr Asp Asn Ala Val Glu Phe Tyr GlnPhe Val Thr Gly Glu 130 135 140 Asp Ala Ala Phe Glu Ser Gly Ala Thr AlaAsp Asp Lys Pro Leu Ser 145 150 155 160 Ser Leu Lys Tyr Val Thr Phe GlyLeu Gly Asn Asn Thr Tyr Glu His 165 170 175 Tyr Asn Ala Met Val Arg AsnVal Asp Ala Ala Leu Thr Lys Phe Gly 180 185 190 Ala Gln Arg Ile Gly SerAla Gly Glu Gly Asp Asp Gly Ala Gly Thr 195 200 205 Met Glu Glu Asp PheLeu Ala Trp Lys Glu Pro Met Trp Ala Ala Leu 210 215 220 Ser Glu Ala MetAsn Leu Gln Glu Arg Asp Ala Val Tyr Glu Pro Val 225 230 235 240 Phe AsnVal Thr Glu Asp Glu Ser Leu Ser Pro Glu Asp Glu Asn Val 245 250 255 TyrLeu Gly Glu Pro Thr Gln Gly His Leu Gln Gly Glu Pro Lys Gly 260 265 270Pro Tyr Ser Ala His Asn Pro Phe Ile Ala Pro Ile Ser Glu Ser Arg 275 280285 Glu Leu Phe Asn Val Lys Asp Arg Asn Cys Leu His Met Glu Ile Ser 290295 300 Ile Ala Gly Ser Asn Leu Thr Tyr Gln Thr Gly Asp His Ile Ala Val305 310 315 320 Trp Pro Thr Asn Ala Gly Ser Glu Val Asp Arg Phe Leu GlnAla Phe 325 330 335 Gly Leu Glu Gly Lys Arg His Ser Val Ile Asn Ile LysGly Ile Asp 340 345 350 Val Thr Ala Lys Val Pro Ile Pro Thr Pro Thr ThrTyr Asp Ala Ala 355 360 365 Val Arg Tyr Tyr Leu Glu Val Cys Ala Pro ValSer Arg Gln Phe Val 370 375 380 Ser Thr Leu Ala Ala Phe Ala Pro Asp GluAla Thr Lys Ala Glu Ile 385 390 395 400 Val Arg Leu Gly Gly Asp Lys AspTyr Phe His Glu Lys Ile Thr Asn 405 410 415 Arg Cys Phe Asn Ile Ala GlnAla Leu Gln Ser Ile Thr Ser Lys Pro 420 425 430 Phe Thr Ala Val Pro PheSer Leu Leu Ile Glu Gly Ile Thr Lys Leu 435 440 445 Gln Pro Arg Tyr TyrSer Ile Ser Ser Ser Ser Leu Val Gln Lys Asp 450 455 460 Lys Ile Ser IleThr Ala Val Val Glu Ser Val Arg Leu Pro Gly Glu 465 470 475 480 Glu HisIle Val Lys Gly Val Thr Thr Asn Tyr Leu Leu Ala Leu Lys 485 490 495 GluLys Gln Asn Gly Glu Pro Ser Pro Asp Pro His Gly Leu Thr Tyr 500 505 510Ser Ile Thr Gly Pro Arg Asn Lys Tyr Asp Gly Ile His Val Pro Val 515 520525 His Val Arg His Ser Asn Phe Lys Leu Pro Ser Asp Pro Ser Arg Pro 530535 540 Val Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly Phe545 550 555 560 Ile Gln Glu Arg Ala Ala Leu Ala Ala Lys Gly Glu Lys ValGly Thr 565 570 575 Thr Leu Leu Phe Phe Gly Cys Arg Lys Ser Asp Glu AspPhe Leu Tyr 580 585 590 Lys Asp Glu Trp Lys Thr Phe Gln Glu Gln Leu GlyAsp Ser Leu Lys 595 600 605 Ile Ile Thr Ala Phe Ser Arg Glu Ser Ala GluLys Val Tyr Val Gln 610 615 620 His Arg Leu Arg Glu His Ala Glu Leu ValSer Asp Leu Leu Lys Gln 625 630 635 640 Lys Ala Thr Phe Tyr Val Cys GlyAsp Ala Ala Asn Met Ala Arg Glu 645 650 655 Val Asn Leu Val Leu Gly GlnIle Ile Ala Lys Gln Arg Gly Leu Pro 660 665 670 Ala Glu Lys Gly Glu GluMet Val Lys His Met Arg Ser Ser Gly Ser 675 680 685 Tyr Gln Asp Asp ValTrp Ser 690 695 7 36 DNA human primer H. oxred 1A 7 gatcggatccaatatgggag actcccacgt ggacac 36 8 36 DNA human primer H. oxred 1B 8gatcggatcc aatatgggag actcccacgt ggacac 36 9 22 DNA human primer H.oxred 2A 9 ctctgctctc gtcaaccagc tg 22 10 35 DNA human primer H. oxred2B 10 gatcggtacc ttagctccac acgtccaggg agtag 35 11 20 DNA Aspergillusprimer A. oxred-for1 modified_base (6)...(6) I - Inosine 11 gacggngcnggtacaatgga 20 12 26 DNA Aspergillus Primer A.oxred-rev1 modified_base(4)...(4) I - Inosine 12 ttangaccan acatcntcct ggtagc 26 13 28 DNA E.coli Primer pSport-for1 13 caagctctaa tacgactcac tataggga 28 14 22 DNAAspergillus Primer A.oxred-rev2 14 caggaaccga tcgacctcgg aa 22 15 25 DNAAspergillus Primer A.oxred-rev3 15 gtcaccctca ccagcagagc caatg 25 16 28DNA Aspergillus Primer A.oxred-rev4 16 ccacattgcg aaccatagcg ttgtagtg 2817 34 DNA E. coli Primer pSport-for2 17 gccaagctct aatacgactc actatagggaaagc 34 18 27 DNA Aspergillus Primer A.oxred-for2 18 gtcgacatggcgcaactcga tactctc 27 19 31 DNA Aspergillus Primer A.oxred-rev5 19ctcgagttag gaccagacat cgtcctggta g 31 20 24 DNA Aspergillus PrimerA.oxred-for3 20 ggatccctcg cgacctgtga tcat 24 21 38 DNA AspergillusPrimer A.oxred-for4 21 cgaagatttc ttgtacaagg atgaatggaa gacttttc 38 2236 DNA Aspergillus Primer A.oxred-rev6 22 ctgaaaagtc ttccattcatccttgtacaa gaaatc 36 23 18 PRT Aspergillus 11aOH peptide 1 23 Ala AlaAla Tyr Trp Leu Ala Thr Leu Gln Pro Ser Asp Leu Pro Glu 1 5 10 15 LeuAsn 24 20 PRT Aspergillus 11aOH peptide 2 24 Cys Arg Gln Ile Leu Thr ProTyr Ile His Lys Arg Lys Ser Leu Lys 1 5 10 15 Gly Thr Thr Asp 20 25 21PRT Aspergillus 11aOH peptide 3 25 His Met Gly Phe Gly His Gly Val HisAla Cys Pro Gly Arg Phe Phe 1 5 10 15 Ala Ser Asn Glu Ile 20 26 20 PRTHuman oxr peptide 1 26 Cys Thr Tyr Trp Ala Val Ala Lys Asp Pro Tyr AlaSer Ala Gly Pro 1 5 10 15 Ala Met Asn Gly 20 27 526 PRT Gibberellafujikuroi CAA75565 27 Met Ala Asn His Ser Ser Ser Tyr Tyr His Glu PheTyr Lys Asp His 1 5 10 15 Ser His Thr Val Leu Thr Leu Met Ser Glu LysPro Val Ile Leu Pro 20 25 30 Ser Leu Ile Leu Gly Thr Cys Ala Val Leu LeuCys Ile Gln Trp Leu 35 40 45 Lys Pro Gln Pro Leu Ile Met Val Asn Gly ArgLys Phe Gly Glu Leu 50 55 60 Ser Asn Val Arg Ala Lys Arg Asp Phe Thr PheGly Ala Arg Gln Leu 65 70 75 80 Leu Glu Lys Gly Leu Lys Met Ser Pro AspLys Pro Phe Arg Ile Met 85 90 95 Gly Asp Val Gly Glu Leu His Ile Leu ProPro Lys Tyr Ala Tyr Glu 100 105 110 Val Arg Asn Asn Glu Lys Leu Ser PheThr Met Ala Ala Phe Lys Trp 115 120 125 Phe Tyr Ala His Leu Pro Gly PheGlu Gly Phe Arg Glu Gly Thr Asn 130 135 140 Glu Ser His Ile Met Lys LeuVal Ala Arg His Gln Leu Thr His Gln 145 150 155 160 Leu Thr Leu Val ThrGly Ala Val Ser Glu Glu Cys Ala Leu Val Leu 165 170 175 Lys Asp Val TyrThr Asp Ser Pro Glu Trp His Asp Ile Thr Ala Lys 180 185 190 Asp Ala AsnMet Lys Leu Met Ala Arg Ile Thr Ser Arg Val Phe Leu 195 200 205 Gly LysGlu Met Cys Arg Asn Pro Gln Trp Leu Arg Ile Thr Ser Thr 210 215 220 TyrAla Val Ile Ala Phe Arg Ala Val Glu Glu Leu Arg Leu Trp Pro 225 230 235240 Ser Trp Leu Arg Pro Val Val Gln Trp Phe Met Pro His Cys Thr Gln 245250 255 Ser Arg Ala Leu Val Gln Glu Ala Arg Asp Leu Ile Asn Pro Leu Leu260 265 270 Glu Arg Arg Arg Glu Glu Lys Ala Glu Ala Glu Arg Thr Gly GluLys 275 280 285 Val Thr Tyr Asn Asp Ala Val Glu Trp Leu Asp Asp Leu AlaArg Glu 290 295 300 Lys Gly Val Gly Tyr Asp Pro Ala Cys Ala Gln Leu SerLeu Ser Val 305 310 315 320 Ala Ala Leu His Ser Thr Thr Asp Phe Phe ThrGln Val Met Phe Asp 325 330 335 Ile Ala Gln Asn Pro Glu Leu Ile Glu ProLeu Arg Glu Glu Ile Ile 340 345 350 Ala Val Leu Gly Lys Gln Gly Trp SerLys Asn Ser Leu Tyr Asn Leu 355 360 365 Lys Leu Met Asp Ser Val Leu LysGlu Ser Gln Arg Leu Lys Pro Ile 370 375 380 Ala Ile Ala Ser Met Arg ArgPhe Thr Thr His Asn Val Lys Leu Ser 385 390 395 400 Asp Gly Val Ile LeuPro Lys Asn Lys Leu Thr Leu Val Ser Ala His 405 410 415 Gln His Trp AspPro Glu Tyr Tyr Lys Asp Pro Leu Lys Phe Asp Gly 420 425 430 Tyr Arg PhePhe Asn Met Arg Arg Glu Pro Gly Lys Glu Ser Lys Ala 435 440 445 Gln LeuVal Ser Ala Thr Pro Asp His Met Gly Phe Gly Tyr Gly Leu 450 455 460 HisAla Cys Pro Gly Arg Phe Phe Ala Ser Glu Glu Ile Lys Ile Ala 465 470 475480 Leu Ser His Ile Leu Leu Lys Tyr Asp Phe Lys Pro Val Glu Gly Ser 485490 495 Ser Met Glu Pro Arg Lys Tyr Gly Leu Asn Met Asn Ala Asn Pro Thr500 505 510 Ala Lys Leu Ser Val Arg Arg Arg Lys Glu Glu Ile Ala Ile 515520 525 28 514 PRT Neurospora crassa CAB91316 28 Met Glu Arg Leu Asp IleLys Ser Ile Thr Asp Pro Ser Ala Thr Pro 1 5 10 15 Phe Ser Tyr Leu ValThr Ala Phe Leu Leu Ala Val Val Val Tyr Ser 20 25 30 Leu Gln Gly Pro ArgPhe Pro Lys Asn Ile Lys His Leu Asn Pro Lys 35 40 45 Gly Pro Leu Glu PheSer Asp Thr Arg Pro Lys Lys Glu Phe Val Tyr 50 55 60 Gly Ser Arg Gln MetLeu Ala Asn Trp Phe Lys Ala Asn Pro Asn Lys 65 70 75 80 Pro Cys Arg ValIle Ser Asp Phe Gly Glu Ala Ile Val Leu Pro Pro 85 90 95 Arg Met Ala AsnGlu Ile Lys Asn Asp Asp Arg Leu Ser Phe Thr Arg 100 105 110 Trp Thr TyrLys Ala Phe His Gly His Leu Pro Gly Phe Glu Gly Phe 115 120 125 Gly GluAla Ser Arg Glu Ser His Ile Val Gln Glu Val Ile Met Arg 130 135 140 AspLeu Thr Lys Tyr Leu Asn Lys Val Thr Glu Pro Leu Ala Gln Glu 145 150 155160 Thr Ser Met Ala Met Glu Ala Asn Leu Pro Lys Ala Ala Asn Gly Glu 165170 175 Trp Ser Thr Ile Asn Leu Arg Ser Lys Ile Leu Pro Ile Val Ala Arg180 185 190 Ile Ser Ser Arg Val Phe Leu Gly Glu Glu Leu Cys Arg Asn GluGlu 195 200 205 Trp Leu Lys Val Thr Gln Gln Tyr Thr Ile Asp Gly Phe GlyAla Ala 210 215 220 Glu Asp Leu Arg Leu Trp Pro Ala Ala Leu Arg Pro IleVal His Trp 225 230 235 240 Phe Leu Pro Ser Cys Gln Arg Ala Arg Ala AspVal Arg Val Ala Arg 245 250 255 Ser Ile Leu Asp Pro Val Leu Lys Lys ArgArg Gln Glu Lys Ala Ala 260 265 270 Asn Gly Gly Lys Ala Glu His Asp AspAla Ile Glu Trp Phe Glu Arg 275 280 285 Thr Ala Lys Gly Lys Tyr Tyr AspPro Ala Val Ala Gln Leu Val Leu 290 295 300 Ser Leu Val Ala Ile His ThrThr Ser Asp Leu Thr Cys Gln Val Met 305 310 315 320 Thr Asn Leu Met GlnAsn Pro Glu Phe Ile Ala Pro Leu Arg Glu Glu 325 330 335 Met Ile Gln ValLeu Ser Glu Gly Gly Trp Lys Lys Thr Ser Leu Tyr 340 345 350 Asn Met LysLeu Leu Asp Ser Val Ile Lys Glu Ser Gln Arg Val Lys 355 360 365 Pro ThrGly Val Ala Ser Met Arg Arg Tyr Ala Glu Lys Asp Val Thr 370 375 380 LeuSer Asp Gly Thr Phe Ile Pro Lys Gly Gly Phe Val Ala Val Ser 385 390 395400 Ala His Asp Met Trp Asn Ser Glu Val Tyr Glu Gln Ala Glu Lys Trp 405410 415 Asp Gly Arg Arg Phe Leu Arg Met Arg Glu Thr Pro Gly Ala Gly Lys420 425 430 Glu Asn Val Ala Gln Leu Val Ser Thr Ala Pro Glu His Leu GlyPhe 435 440 445 Gly His Gly Gln His Ala Cys Pro Gly Arg Phe Phe Ala AlaAsn Glu 450 455 460 Ile Lys Ile Ala Leu Val His Leu Leu Leu Asn Tyr GluTrp Arg Leu 465 470 475 480 Pro Glu Gly Ser Asp Pro Lys Ile Arg Thr PheGly Phe Ser Met Gly 485 490 495 Val Asp Pro Ser Leu Lys Val Glu Tyr LysGly Arg Gln Pro Glu Ile 500 505 510 Glu Leu 29 495 PRT Catharanthusroseus CAB56503 29 Leu Leu Phe Cys Phe Ile Leu Ser Lys Thr Thr Lys LysPhe Gly Gln 1 5 10 15 Asn Ser Gln Tyr Ser Asn His Asp Glu Leu Pro ProGly Pro Pro Gln 20 25 30 Ile Pro Ile Leu Gly Asn Ala His Gln Leu Ser GlyGly His Thr His 35 40 45 His Ile Leu Arg Asp Leu Ala Lys Lys Tyr Gly ProLeu Met His Leu 50 55 60 Lys Ile Gly Glu Val Ser Thr Ile Val Ala Ser SerPro Gln Ile Ala 65 70 75 80 Glu Glu Ile Phe Arg Thr His Asp Ile Leu PheAla Asp Arg Pro Ser 85 90 95 Asn Leu Glu Ser Phe Lys Ile Val Ser Tyr AspPhe Ser Asp Met Val 100 105 110 Val Ser Pro Tyr Gly Asn Tyr Trp Arg GlnLeu Arg Lys Ile Ser Met 115 120 125 Met Glu Leu Leu Ser Gln Lys Ser ValGln Ser Phe Arg Ser Ile Arg 130 135 140 Glu Glu Glu Val Leu Asn Phe IleLys Ser Ile Gly Ser Lys Glu Gly 145 150 155 160 Thr Arg Ile Asn Leu SerLys Glu Ile Ser Leu Leu Ile Tyr Gly Ile 165 170 175 Thr Thr Arg Ala AlaPhe Gly Glu Lys Asn Lys Asn Thr Glu Glu Phe 180 185 190 Ile Arg Leu LeuAsp Gln Leu Thr Lys Ala Val Ala Glu Pro Asn Ile 195 200 205 Ala Asp MetPhe Pro Ser Leu Lys Phe Leu Gln Leu Ile Ser Thr Ser 210 215 220 Lys TyrLys Ile Glu Lys Ile His Lys Gln Phe Asp Val Ile Val Glu 225 230 235 240Thr Ile Leu Lys Gly His Lys Glu Lys Ile Asn Lys Pro Leu Ser Gln 245 250255 Glu Asn Gly Glu Lys Lys Glu Asp Leu Val Asp Val Leu Leu Asn Ile 260265 270 Gln Arg Arg Asn Asp Phe Glu Ala Pro Leu Gly Asp Lys Asn Ile Lys275 280 285 Ala Ile Ile Phe Asn Ile Phe Ser Ala Gly Thr Glu Thr Ser SerThr 290 295 300 Thr Val Asp Trp Ala Met Cys Glu Met Ile Lys Asn Pro ThrVal Met 305 310 315 320 Lys Lys Ala Gln Glu Glu Val Arg Lys Val Phe AsnGlu Glu Gly Asn 325 330 335 Val Asp Glu Thr Lys Leu His Gln Leu Lys TyrLeu Gln Ala Val Ile 340 345 350 Lys Glu Thr Leu Arg Leu His Pro Pro ValPro Leu Leu Leu Pro Arg 355 360 365 Glu Cys Arg Glu Gln Cys Lys Ile LysGly Tyr Thr Ile Pro Ser Lys 370 375 380 Ser Arg Val Ile Val Asn Ala TrpAla Ile Gly Arg Asp Pro Asn Tyr 385 390 395 400 Trp Ile Glu Pro Glu LysPhe Asn Pro Asp Arg Phe Leu Glu Ser Lys 405 410 415 Val Asp Phe Lys GlyAsn Ser Phe Glu Tyr Leu Pro Phe Gly Gly Gly 420 425 430 Arg Arg Ile CysPro Gly Ile Thr Phe Ala Leu Ala Asn Ile Glu Leu 435 440 445 Pro Leu AlaGln Leu Leu Phe His Phe Asp Trp Gln Ser Asn Thr Glu 450 455 460 Lys LeuAsn Met Lys Glu Ser Arg Gly Val Thr Val Arg Arg Glu Asp 465 470 475 480Asp Leu Tyr Leu Thr Pro Val Asn Phe Ser Ser Ser Ser Pro Ala 485 490 49530 510 PRT Glycine max AAB94588 30 Met Val Met Glu Leu His Asn His ThrPro Phe Ser Ile Tyr Phe Ile 1 5 10 15 Thr Ser Ile Leu Phe Ile Phe PheVal Phe Phe Lys Leu Val Gln Arg 20 25 30 Ser Asp Ser Lys Thr Ser Ser ThrCys Lys Leu Pro Pro Gly Pro Arg 35 40 45 Thr Leu Pro Leu Ile Gly Asn IleHis Gln Ile Val Gly Ser Leu Pro 50 55 60 Val His Tyr Tyr Leu Lys Asn LeuAla Asp Lys Tyr Gly Pro Leu Met 65 70 75 80 His Leu Lys Leu Gly Glu ValSer Asn Ile Ile Val Thr Ser Pro Glu 85 90 95 Met Ala Gln Glu Ile Met LysThr His Asp Leu Asn Phe Ser Asp Arg 100 105 110 Pro Asp Phe Val Leu SerArg Ile Val Ser Tyr Asn Gly Ser Gly Ile 115 120 125 Val Phe Ser Gln HisGly Asp Tyr Trp Arg Gln Leu Arg Lys Ile Cys 130 135 140 Thr Val Glu LeuLeu Thr Ala Lys Arg Val Gln Ser Phe Arg Ser Ile 145 150 155 160 Arg GluGlu Glu Val Ala Glu Leu Val Lys Lys Ile Ala Ala Thr Ala 165 170 175 SerGlu Glu Gly Gly Ser Ile Phe Asn Leu Thr Gln Ser Ile Tyr Ser 180 185 190Met Thr Phe Gly Ile Ala Ala Arg Ala Ala Phe Gly Lys Lys Ser Arg 195 200205 Tyr Gln Gln Val Phe Ile Ser Asn Met His Lys Gln Leu Met Leu Leu 210215 220 Gly Gly Phe Ser Val Ala Asp Leu Tyr Pro Ser Ser Arg Val Phe Gln225 230 235 240 Met Met Gly Ala Thr Gly Lys Leu Glu Lys Val His Arg ValThr Asp 245 250 255 Arg Val Leu Gln Asp Ile Ile Asp Glu His Lys Asn ArgAsn Arg Ser 260 265 270 Ser Glu Glu Arg Glu Ala Val Glu Asp Leu Val AspVal Leu Leu Lys 275 280 285 Phe Gln Lys Glu Ser Glu Phe Arg Leu Thr AspAsp Asn Ile Lys Ala 290 295 300 Val Ile Gln Asp Ile Phe Ile Gly Gly GlyGlu Thr Ser Ser Ser Val 305 310 315 320 Val Glu Trp Gly Met Ser Glu LeuIle Arg Asn Pro Arg Val Met Glu 325 330 335 Glu Ala Gln Ala Glu Val ArgArg Val Tyr Asp Ser Lys Gly Tyr Val 340 345 350 Asp Glu Thr Glu Leu HisGln Leu Ile Tyr Leu Lys Ser Ile Ile Lys 355 360 365 Glu Thr Met Arg LeuHis Pro Pro Val Pro Leu Leu Val Pro Arg Val 370 375 380 Ser Arg Glu ArgCys Gln Ile Asn Gly Tyr Glu Ile Pro Ser Lys Thr 385 390 395 400 Arg IleIle Ile Asn Ala Trp Ala Ile Gly Arg Asn Pro Lys Tyr Trp 405 410 415 GlyGlu Thr Glu Ser Phe Lys Pro Glu Arg Phe Leu Asn Ser Ser Ile 420 425 430Asp Phe Arg Gly Thr Asp Phe Glu Phe Ile Pro Phe Gly Ala Gly Arg 435 440445 Arg Ile Cys Pro Gly Ile Thr Phe Ala Ile Pro Asn Ile Glu Leu Pro 450455 460 Leu Ala Gln Leu Leu Tyr His Phe Asp Trp Lys Leu Pro Asn Lys Met465 470 475 480 Lys Asn Glu Glu Leu Asp Met Thr Glu Ser Asn Gly Ile ThrLeu Arg 485 490 495 Arg Gln Asn Asp Leu Cys Leu Ile Pro Ile Thr Arg LeuPro 500 505 510 31 524 PRT Gibberella fujikuroi CAA75566 31 Met Ser IlePhe Asn Met Ile Thr Ser Tyr Ala Gly Ser Gln Leu Leu 1 5 10 15 Pro PheTyr Ile Ala Ile Phe Val Phe Thr Leu Val Pro Trp Ala Ile 20 25 30 Arg PheSer Trp Leu Glu Leu Arg Lys Gly Ser Val Val Pro Leu Ala 35 40 45 Asn ProPro Asp Ser Leu Phe Gly Thr Gly Lys Thr Arg Arg Ser Phe 50 55 60 Val LysLeu Ser Arg Glu Ile Leu Ala Lys Ala Arg Ser Leu Phe Pro 65 70 75 80 AsnGlu Pro Phe Arg Leu Ile Thr Asp Trp Gly Glu Val Leu Ile Leu 85 90 95 ProPro Asp Phe Ala Asp Glu Ile Arg Asn Asp Pro Arg Leu Ser Phe 100 105 110Ser Lys Ala Ala Met Gln Asp Asn His Ala Gly Ile Pro Gly Phe Glu 115 120125 Thr Val Ala Leu Val Gly Arg Glu Asp Gln Leu Ile Gln Lys Val Ala 130135 140 Arg Lys Gln Leu Thr Lys His Leu Ser Ala Val Ile Glu Pro Leu Ser145 150 155 160 Arg Glu Ser Thr Leu Ala Val Ser Leu Asn Phe Gly Glu ThrThr Glu 165 170 175 Trp Arg Ala Ile Arg Leu Lys Pro Ala Ile Leu Asp IleIle Ala Arg 180 185 190 Ile Ser Ser Arg Ile Tyr Leu Gly Asp Gln Leu CysArg Asn Glu Ala 195 200 205 Trp Leu Lys Ile Thr Lys Thr Tyr Thr Thr AsnPhe Tyr Thr Ala Ser 210 215 220 Thr Asn Leu Arg Met Phe Pro Arg Ser IleArg Pro Leu Ala His Trp 225 230 235 240 Phe Leu Pro Glu Cys Arg Lys LeuArg Gln Glu Arg Lys Asp Ala Ile 245 250 255 Gly Ile Ile Thr Pro Leu IleGlu Arg Arg Arg Glu Leu Arg Arg Ala 260 265 270 Ala Ile Ala Ala Gly GlnPro Leu Pro Val Phe His Asp Ala Ile Asp 275 280 285 Trp Ser Glu Gln GluAla Glu Ala Ala Gly Thr Gly Ala Ser Phe Asp 290 295 300 Pro Val Ile PheGln Leu Thr Leu Ser Leu Leu Ala Ile His Thr Thr 305 310 315 320 Tyr AspLeu Leu Gln Gln Thr Met Ile Asp Leu Gly Arg His Pro Glu 325 330 335 TyrIle Glu Pro Leu Arg Gln Glu Val Val Gln Leu Leu Arg Glu Glu 340 345 350Gly Trp Lys Lys Thr Thr Leu Phe Lys Met Lys Leu Leu Asp Ser Ala 355 360365 Ile Lys Glu Ser Gln Arg Met Lys Pro Gly Ser Ile Val Thr Met Arg 370375 380 Arg Tyr Val Thr Glu Asp Ile Thr Leu Ser Ser Gly Leu Thr Leu Lys385 390 395 400 Lys Gly Thr Arg Leu Asn Val Asp Asn Arg Arg Leu Asp AspPro Lys 405 410 415 Ile Tyr Asp Asn Pro Glu Val Tyr Asn Pro Tyr Arg PheTyr Asp Met 420 425 430 Arg Ser Glu Ala Gly Lys Asp His Gly Ala Gln LeuVal Ser Thr Gly 435 440 445 Ser Asn His Met Gly Phe Gly His Gly Gln HisSer Cys Pro Gly Arg 450 455 460 Phe Phe Ala Ala Asn Glu Ile Lys Val AlaLeu Cys His Ile Leu Val 465 470 475 480 Lys Tyr Asp Trp Lys Leu Cys ProAsp Thr Glu Thr Lys Pro Asp Thr 485 490 495 Arg Gly Met Ile Ala Lys SerSer Pro Val Thr Asp Ile Leu Ile Lys 500 505 510 Arg Arg Glu Ser Val GluLeu Asp Leu Glu Ala Ile 515 520 32 528 PRT Aspergillus terreus AAD3455232 Met Thr Val Asp Ala Leu Thr Gln Pro His His Leu Leu Ser Leu Ala 1 510 15 Trp Asn Asp Thr Gln Gln His Gly Ser Trp Phe Ala Pro Leu Val Thr 2025 30 Thr Ser Ala Gly Leu Leu Cys Leu Leu Leu Tyr Leu Cys Ser Ser Gly 3540 45 Arg Arg Ser Asp Leu Pro Val Phe Asn Pro Lys Thr Trp Trp Glu Leu 5055 60 Thr Thr Met Arg Ala Lys Arg Asp Phe Asp Ala Asn Ala Pro Ser Trp 6570 75 80 Ile Glu Ser Trp Phe Ser Gln Asn Asp Lys Pro Ile Arg Phe Ile Val85 90 95 Asp Ser Gly Tyr Cys Thr Ile Leu Pro Ser Ser Met Ala Asp Glu Phe100 105 110 Arg Lys Met Lys Glu Leu Cys Met Tyr Lys Phe Leu Gly Thr AspPhe 115 120 125 His Ser His Leu Pro Gly Phe Asp Gly Phe Lys Glu Val ThrArg Asp 130 135 140 Ala His Leu Ile Thr Lys Val Val Met Asn Gln Phe GlnThr Gln Ala 145 150 155 160 Pro Lys Tyr Val Lys Pro Leu Ala Asn Glu AlaSer Gly Ile Ile Thr 165 170 175 Asp Ile Phe Gly Asp Ser Asn Glu Trp HisThr Val Pro Val Tyr Asn 180 185 190 Gln Cys Leu Asp Leu Val Thr Arg ThrVal Thr Phe Ile Met Val Gly 195 200 205 Ser Lys Leu Ala His Asn Glu GluTrp Leu Asp Ile Ala Lys His His 210 215 220 Ala Val Thr Met Ala Ile GlnAla Arg Gln Leu Arg Leu Trp Pro Val 225 230 235 240 Ile Leu Arg Pro LeuVal His Trp Leu Glu Pro Gln Gly Ala Lys Leu 245 250 255 Arg Ala Gln ValArg Arg Ala Arg Gln Leu Leu Asp Pro Ile Ile Gln 260 265 270 Glu Arg ArgAla Glu Arg Asp Ala Cys Arg Ala Lys Gly Ile Glu Pro 275 280 285 Pro ArgTyr Val Asp Ser Ile Gln Trp Phe Glu Asp Thr Ala Lys Gly 290 295 300 LysTrp Tyr Asp Ala Ala Gly Ala Gln Leu Ala Met Asp Phe Ala Gly 305 310 315320 Ile Tyr Gly Thr Ser Asp Leu Leu Ile Gly Gly Leu Val Asp Ile Val 325330 335 Arg His Pro His Leu Leu Glu Pro Leu Arg Asp Glu Ile Arg Thr Val340 345 350 Ile Gly Gln Gly Gly Trp Thr Pro Ala Ser Leu Tyr Lys Leu LysLeu 355 360 365 Leu Asp Ser Cys Leu Lys Glu Ser Gln Arg Val Lys Pro ValGlu Cys 370 375 380 Ala Thr Met Arg Ser Tyr Ala Leu Gln Asp Val Thr PheSer Asn Gly 385 390 395 400 Thr Phe Ile Pro Lys Gly Glu Leu Val Ala ValAla Ala Asp Arg Met 405 410 415 Ser Asn Pro Glu Val Trp Pro Glu Pro AlaLys Tyr Asp Pro Tyr Arg 420 425 430 Tyr Met Arg Leu Arg Glu Asp Pro AlaLys Ala Phe Ser Ala Gln Leu 435 440 445 Glu Asn Thr Asn Gly Asp His IleGly Phe Gly Trp His Pro Arg Ala 450 455 460 Cys Pro Gly Arg Phe Phe AlaSer Lys Glu Ile Lys Met Met Leu Ala 465 470 475 480 Tyr Leu Leu Ile ArgTyr Asp Trp Lys Val Val Pro Asp Glu Pro Leu 485 490 495 Gln Tyr Tyr ArgHis Ser Phe Ser Val Arg Ile His Pro Thr Thr Lys 500 505 510 Leu Met MetArg Arg Arg Asp Glu Asp Ile Arg Leu Pro Gly Ser Leu 515 520 525 33 388PRT Gibberella fujikuroi CAA75567 33 Met Lys Tyr Thr Thr Cys Gln Met AsnIle Phe Pro Ser Leu Trp Ser 1 5 10 15 Met Lys Thr Ser Phe Arg Trp ProArg Thr Ser Lys Trp Ser Ser Val 20 25 30 Ser Leu Tyr Asp Met Met Leu ArgThr Val Ala Leu Leu Ser Gly Arg 35 40 45 Ala Phe Val Gly Leu Pro Leu CysArg Asp Glu Gly Trp Leu Gln Ala 50 55 60 Ser Ile Gly Tyr Thr Val Gln CysVal Ser Ile Arg Asp Gln Leu Phe 65 70 75 80 Thr Trp Ser Pro Val Leu ArgPro Ile Ile Gly Pro Phe Leu Pro Ser 85 90 95 Val Arg Ser Val Arg Arg HisLeu Arg Phe Ala Ala Glu Ile Met Ala 100 105 110 Pro Leu Ile Ser Gln AlaLeu Gln Asp Glu Lys Gln His Arg Ala Asp 115 120 125 Thr Leu Leu Ala AspGln Thr Glu Gly Arg Gly Thr Phe Ile Ser Trp 130 135 140 Leu Leu Arg HisLeu Pro Glu Glu Leu Arg Thr Pro Glu Gln Val Gly 145 150 155 160 Leu AspGln Met Leu Val Ser Phe Ala Ala Ile His Thr Thr Thr Met 165 170 175 AlaLeu Thr Lys Val Val Trp Glu Leu Val Lys Arg Pro Glu Tyr Ile 180 185 190Glu Pro Leu Arg Thr Glu Met Gln Asp Val Phe Gly Pro Asp Ala Val 195 200205 Ser Pro Asp Ile Cys Ile Asn Lys Glu Ala Leu Ser Arg Leu His Lys 210215 220 Leu Asp Ser Phe Ile Arg Glu Val Gln Arg Trp Cys Pro Ser Thr Phe225 230 235 240 Val Thr Pro Ser Arg Arg Val Met Lys Ser Met Thr Leu SerAsn Gly 245 250 255 Ile Lys Leu Gln Arg Gly Thr Ser Ile Ala Phe Pro AlaHis Ala Ile 260 265 270 His Met Ser Glu Glu Thr Pro Thr Phe Ser Pro AspPhe Ser Ser Asp 275 280 285 Phe Glu Asn Pro Ser Pro Arg Ile Phe Asp GlyPhe Arg Tyr Leu Asn 290 295 300 Leu Arg Ser Ile Lys Gly Gln Gly Ser GlnHis Gln Ala Ala Thr Thr 305 310 315 320 Gly Pro Asp Tyr Leu Ile Phe AsnHis Gly Lys His Ala Cys Pro Gly 325 330 335 Arg Phe Phe Ala Ile Ser GluIle Lys Met Ile Leu Ile Glu Leu Leu 340 345 350 Ala Lys Tyr Asp Phe ArgLeu Glu Asp Gly Lys Pro Gly Pro Glu Leu 355 360 365 Met Arg Val Gly ThrGlu Thr Arg Leu Asp Thr Lys Ala Gly Leu Glu 370 375 380 Met Arg Arg Arg385 34 525 PRT Gibberella fujikuroi CAA76703 34 Met Ser Lys Ser Asn SerMet Asn Ser Thr Ser His Glu Thr Leu Phe 1 5 10 15 Gln Gln Leu Val LeuGly Leu Asp Arg Met Pro Leu Met Asp Val His 20 25 30 Trp Leu Ile Tyr ValAla Phe Gly Ala Trp Leu Cys Ser Tyr Val Ile 35 40 45 His Val Leu Ser SerSer Ser Thr Val Lys Val Pro Val Val Gly Tyr 50 55 60 Arg Ser Val Phe GluPro Thr Trp Leu Leu Arg Leu Arg Phe Val Trp 65 70 75 80 Glu Gly Gly SerIle Ile Gly Gln Gly Tyr Asn Lys Phe Lys Asp Ser 85 90 95 Ile Phe Gln ValArg Lys Leu Gly Thr Asp Ile Val Ile Ile Pro Pro 100 105 110 Asn Tyr IleAsp Glu Val Arg Lys Leu Ser Gln Asp Lys Thr Arg Ser 115 120 125 Val GluPro Phe Ile Asn Asp Phe Ala Gly Gln Tyr Thr Arg Gly Met 130 135 140 ValPhe Leu Gln Ser Asp Leu Gln Asn Arg Val Ile Gln Gln Arg Leu 145 150 155160 Thr Pro Lys Leu Val Ser Leu Thr Lys Val Met Lys Glu Glu Leu Asp 165170 175 Tyr Ala Leu Thr Lys Glu Met Pro Asp Met Lys Asn Asp Glu Trp Val180 185 190 Glu Val Asp Ile Ser Ser Ile Met Val Arg Leu Ile Ser Arg IleSer 195 200 205 Ala Arg Val Phe Leu Gly Pro Glu His Cys Arg Asn Gln GluTrp Leu 210 215 220 Thr Thr Thr Ala Glu Tyr Ser Glu Ser Leu Phe Ile ThrGly Phe Ile 225 230 235 240 Leu Arg Val Val Pro His Ile Leu Arg Pro PheIle Ala Pro Leu Leu 245 250 255 Pro Ser Tyr Arg Thr Leu Leu Arg Asn ValSer Ser Gly Arg Arg Val 260 265 270 Ile Gly Asp Ile Ile Arg Ser Gln GlnGly Asp Gly Asn Glu Asp Ile 275 280 285 Leu Ser Trp Met Arg Asp Ala AlaThr Gly Glu Glu Lys Gln Ile Asp 290 295 300 Asn Ile Ala Gln Arg Met LeuIle Leu Ser Leu Ala Ser Ile His Thr 305 310 315 320 Thr Ala Met Thr MetThr His Ala Met Tyr Asp Leu Cys Ala Cys Pro 325 330 335 Glu Tyr Ile GluPro Leu Arg Asp Glu Val Lys Ser Val Val Gly Ala 340 345 350 Ser Gly TrpAsp Lys Thr Ala Leu Asn Arg Phe His Lys Leu Asp Ser 355 360 365 Phe LeuLys Glu Ser Gln Arg Phe Asn Pro Val Phe Leu Leu Thr Phe 370 375 380 AsnArg Ile Tyr His Gln Ser Met Thr Leu Ser Asp Gly Thr Asn Ile 385 390 395400 Pro Ser Gly Thr Arg Ile Ala Val Pro Ser His Ala Met Leu Gln Asp 405410 415 Ser Ala His Val Pro Gly Pro Thr Pro Pro Thr Glu Phe Asp Gly Phe420 425 430 Arg Tyr Ser Lys Ile Arg Ser Asp Ser Asn Tyr Ala Gln Lys TyrLeu 435 440 445 Phe Ser Met Thr Asp Ser Ser Asn Met Ala Phe Gly Tyr GlyLys Tyr 450 455 460 Ala Cys Pro Gly Arg Phe Tyr Ala Ser Asn Glu Met LysLeu Thr Leu 465 470 475 480 Ala Ile Leu Leu Leu Gln Phe Glu Phe Lys LeuPro Asp Gly Lys Gly 485 490 495 Arg Pro Arg Asn Ile Thr Ile Asp Ser AspMet Ile Pro Asp Pro Arg 500 505 510 Ala Arg Leu Cys Val Arg Lys Arg SerLeu Arg Asp Glu 515 520 525 35 294 PRT Fusarium oxysporum CAA57874 35Met Ala Pro Met Leu Arg Pro Leu Val Tyr Arg Phe Ile Pro Glu Arg 1 5 1015 Ala Arg Ile Lys Asp Gln Trp Thr Lys Gly Arg Lys Arg Val Met Ala 20 2530 Ser Met Arg Glu Arg Gln Glu Lys Gly Gly Asn Leu Glu Asp Pro Pro 35 4045 Thr Met Leu Asp His Leu Ser Asn Gly Arg Asn Glu His Ile Ala Asp 50 5560 Asp Val Glu Leu Gln Leu Leu His Gln Met Thr Leu Ile Ala Val Gly 65 7075 80 Thr Val Thr Thr Phe Ser Ser Thr Thr Gln Ala Ile Tyr Asp Leu Val 8590 95 Ala His Pro Glu Tyr Ile Thr Ile Leu Arg Glu Glu Val Glu Ser Val100 105 110 Pro Arg Asp Pro Asn Gly Asn Phe Thr Lys Asp Ser Thr Val AlaMet 115 120 125 Asp Lys Leu Asp Ser Phe Leu Lys Glu Ser Gln Arg Phe AsnSer Pro 130 135 140 Asp Leu Ser Met Ser Asn Leu Lys Asn Tyr Lys Leu CysGlu Ser Leu 145 150 155 160 Thr Gly His Ser Asn Leu Pro Thr Arg Thr IleAla Asp Met Lys Leu 165 170 175 Pro Asp Gly Thr Phe Val Pro Lys Gly ThrLys Leu Glu Ile Asn Thr 180 185 190 Cys Ser Ile His Lys Asp His Lys LeuTyr Glu Asn Pro Glu Gln Phe 195 200 205 Asp Gly Leu Arg Phe His Lys TrpArg Lys Ala Pro Gly Lys Glu Lys 210 215 220 Arg Tyr Met Tyr Ser Ser SerGly Thr Asp Asp Leu Ser Trp Gly Phe 225 230 235 240 Gly Arg His Ala CysPro Gly Arg Tyr Leu Ser Ala Ile Asn Ile Lys 245 250 255 Leu Ile Met AlaGlu Leu Leu Met Asn Tyr Asp Ile Lys Leu Pro Asp 260 265 270 Gly Leu SerArg Pro Lys Asn Ile Glu Phe Glu Val Leu Ala Ser Leu 275 280 285 Asn AlaCys Ala Asn Ala 290 36 510 PRT Caenorhabditis elegans CAA91268 36 MetAla Leu Leu Ile Leu Ser Ser Leu Val Ile Ser Ile Phe Thr Phe 1 5 10 15Phe Ile Tyr Ile Ile Leu Ala Arg Arg Glu Arg Phe Lys Leu Arg Glu 20 25 30Lys Ile Gly Leu Ser Gly Pro Glu Pro His Trp Phe Leu Gly Asn Leu 35 40 45Lys Gln Thr Ala Glu Arg Lys Glu Lys Leu Gly Tyr Asp Asp Ala Asn 50 55 60Arg Trp Phe Asn Glu Leu His Glu Gln Tyr Gly Glu Thr Phe Gly Ile 65 70 7580 Tyr Tyr Gly Ser Gln Met Asn Ile Val Ile Ser Asn Glu Lys Asp Ile 85 9095 Lys Glu Val Phe Ile Lys Asn Phe Ser Asn Phe Ser Asp Arg Ser Val 100105 110 Pro Ser Ile Tyr Glu Ala Asn Gln Leu Thr Ala Ser Leu Leu Met Asn115 120 125 Ser Tyr Ser Ser Gly Trp Lys His Thr Arg Ser Ala Ile Ala ProIle 130 135 140 Phe Ser Thr Gly Lys Met Lys Ala Met Gln Glu Thr Ile AsnSer Lys 145 150 155 160 Val Asp Leu Phe Leu Asp Ile Leu Arg Glu Lys AlaSer Ser Gly Gln 165 170 175 Lys Trp Asp Ile Tyr Asp Asp Phe Gln Gly LeuThr Leu Asp Val Ile 180 185 190 Gly Lys Cys Ala Phe Ala Ile Asp Ser AsnCys Gln Arg Asp Arg Asn 195 200 205 Asp Val Phe Tyr His Pro Val Thr ValLys Ile Thr Ile Asn Asn Phe 210 215 220 Thr Tyr Phe His Ser Ser Ser ProGly Thr Phe His Phe Leu Glu Ser 225 230 235 240 Thr Leu Gln Ile His ThrThr Gly Arg Cys Arg Asn Ser Thr Cys Arg 245 250 255 Arg Thr Val Lys CysVal Gly Phe Arg Gln Asp Lys Ala Lys Phe Cys 260 265 270 Ser Asp Tyr GluArg Arg Arg Gly Gly Glu Gly Ser Asp Ser Val Asp 275 280 285 Leu Leu LysLeu Leu Leu Asn Arg Glu Asp Asp Lys Ser Lys Pro Met 290 295 300 Thr LysGln Glu Val Ile Glu Asn Cys Phe Ala Phe Leu Leu Ala Gly 305 310 315 320Tyr Glu Thr Thr Ser Thr Ala Met Thr Tyr Cys Ser Tyr Leu Leu Ser 325 330335 Lys Tyr Pro Asn Val Gln Gln Lys Leu Tyr Glu Glu Ile Met Glu Ala 340345 350 Lys Glu Asn Gly Gly Leu Thr Tyr Asp Ser Ile His Asn Met Lys Tyr355 360 365 Leu Asp Cys Val Tyr Lys Glu Thr Leu Arg Phe Tyr Pro Pro HisPhe 370 375 380 Ser Phe Ile Arg Arg Leu Cys Arg Glu Asp Ile Thr Ile ArgGly Gln 385 390 395 400 Phe Tyr Pro Lys Gly Ala Ile Val Val Cys Leu ProHis Thr Val His 405 410 415 Arg Asn Pro Glu Asn Trp Asp Ser Pro Glu GluPhe His Pro Glu Arg 420 425 430 Phe Glu Asn Trp Glu Glu Lys Ser Ser SerLeu Lys Trp Ile Pro Phe 435 440 445 Gly Val Gly Pro Arg Tyr Cys Val GlyMet Arg Phe Ala Glu Met Glu 450 455 460 Phe Lys Thr Thr Ile Val Lys LeuLeu Asp Thr Phe Glu Leu Lys Gln 465 470 475 480 Phe Glu Gly Glu Ala AspLeu Ile Pro Asp Cys Asn Gly Val Ile Met 485 490 495 Arg Pro Asn Asp ProVal Arg Leu His Leu Lys Pro Arg Asn 500 505 510 37 691 PRT yeast P450reductase 37 Met Pro Phe Gly Ile Asp Asn Thr Asp Phe Thr Val Leu Ala GlyLeu 1 5 10 15 Val Leu Ala Val Leu Leu Tyr Val Lys Arg Asn Ser Ile LysGlu Leu 20 25 30 Leu Met Ser Asp Asp Gly Asp Ile Thr Ala Val Ser Ser GlyAsn Arg 35 40 45 Asp Ile Ala Gln Val Val Thr Glu Asn Asn Lys Asn Tyr LeuVal Leu 50 55 60 Tyr Ala Ser Gln Thr Gly Thr Ala Glu Asp Tyr Ala Lys LysPhe Ser 65 70 75 80 Lys Glu Leu Val Ala Lys Phe Asn Leu Asn Val Met CysAla Asp Val 85 90 95 Glu Asn Tyr Asp Phe Glu Ser Leu Asn Asp Val Pro ValIle Val Ser 100 105 110 Ile Phe Ile Ser Thr Tyr Gly Glu Gly Asp Phe ProAsp Gly Ala Val 115 120 125 Asn Phe Glu Asp Phe Ile Cys Asn Ala Glu AlaGly Ala Leu Ser Asn 130 135 140 Leu Arg Tyr Asn Met Phe Gly Leu Gly AsnSer Thr Tyr Glu Phe Phe 145 150 155 160 Asn Gly Ala Ala Lys Lys Ala GluLys His Leu Ser Ala Ala Gly Ala 165 170 175 Ile Arg Leu Gly Lys Leu GlyGlu Ala Asp Asp Gly Ala Gly Thr Thr 180 185 190 Asp Glu Asp Tyr Met AlaTrp Lys Asp Ser Ile Leu Glu Val Leu Lys 195 200 205 Asp Glu Leu His LeuAsp Glu Gln Glu Ala Lys Phe Thr Ser Gln Phe 210 215 220 Gln Tyr Thr ValLeu Asn Glu Ile Thr Asp Ser Met Ser Leu Gly Glu 225 230 235 240 Pro SerAla His Tyr Leu Pro Ser His Gln Leu Asn Arg Asn Ala Asp 245 250 255 GlyIle Gln Leu Gly Pro Phe Asp Leu Ser Gln Pro Tyr Ile Ala Pro 260 265 270Ile Val Lys Ser Arg Glu Leu Phe Ser Ser Asn Asp Arg Asn Cys Ile 275 280285 His Ser Glu Phe Asp Leu Ser Gly Ser Asn Ile Lys Tyr Ser Thr Gly 290295 300 Asp His Leu Ala Val Trp Pro Ser Asn Pro Leu Glu Lys Val Glu Gln305 310 315 320 Phe Leu Ser Ile Phe Asn Leu Asp Pro Glu Thr Ile Phe AspLeu Lys 325 330 335 Pro Leu Asp Pro Thr Val Lys Val Pro Phe Pro Thr ProThr Thr Ile 340 345 350 Gly Ala Ala Ile Lys His Tyr Leu Glu Ile Thr GlyPro Val Ser Arg 355 360 365 Gln Leu Phe Ser Ser Leu Ile Gln Phe Ala ProAsn Ala Asp Val Lys 370 375 380 Glu Lys Leu Thr Leu Leu Ser Lys Asp LysAsp Gln Phe Ala Val Glu 385 390 395 400 Ile Thr Ser Lys Tyr Phe Asn IleAla Asp Ala Leu Lys Tyr Leu Ser 405 410 415 Asp Gly Ala Lys Trp Asp AsnVal Pro Met Gln Phe Leu Val Glu Ser 420 425 430 Val Pro Gln Met Thr ProArg Tyr Tyr Ser Ile Ser Ser Ser Ser Leu 435 440 445 Ser Glu Lys Gln ThrVal His Val Thr Ser Ile Val Glu Asn Phe Pro 450 455 460 Asn Pro Glu LeuPro Asp Ala Pro Pro Gly Val Gly Val Thr Thr Asn 465 470 475 480 Leu LeuArg Asn Ile Gln Leu Ala Gln Asn Asn Val Asn Ile Ala Glu 485 490 495 ThrAsn Leu Pro Val His Tyr Asp Leu Asn Gly Pro Arg Lys Leu Phe 500 505 510Ala Asn Tyr Lys Leu Pro Val His Val Arg Arg Ser Asn Phe Arg Leu 515 520525 Pro Ser Asn Pro Ser Thr Pro Val Ile Met Ile Gly Pro Gly Thr Gly 530535 540 Val Ala Pro Phe Arg Gly Phe Ile Arg Glu Arg Val Ala Phe Leu Glu545 550 555 560 Ser Gln Lys Lys Gly Gly Asn Asn Val Ser Leu Gly Lys HisIle Leu 565 570 575 Phe Tyr Gly Ser Arg Asn Thr Asp Asp Phe Leu Tyr GlnAsp Glu Trp 580 585 590 Pro Glu Tyr Ala Lys Lys Leu Asp Gly Ser Phe GluMet Val Val Ala 595 600 605 His Ser Arg Leu Pro Asn Thr Lys Lys Val TyrVal Gln Asp Lys Leu 610 615 620 Lys Asp Tyr Glu Asp Gln Val Phe Glu MetIle Asn Asn Gly Ala Phe 625 630 635 640 Ile Tyr Val Cys Gly Asp Ala LysGly Met Ala Lys Gly Val Ser Thr 645 650 655 Ala Leu Val Gly Ile Leu SerArg Gly Lys Ser Ile Thr Thr Asp Glu 660 665 670 Ala Thr Glu Leu Ile LysMet Leu Lys Thr Ser Gly Arg Tyr Gln Glu 675 680 685 Asp Val Trp 690 38693 PRT Aspergillus niger P450 reductase 38 Met Ala Gln Leu Asp Thr LeuAsp Leu Val Val Leu Ala Val Leu Leu 1 5 10 15 Val Gly Ser Val Ala TyrPhe Thr Lys Gly Thr Tyr Trp Ala Val Ala 20 25 30 Lys Thr Arg Met Pro LeuPro Ala Pro Arg Met Asn Gly Ala Ala Lys 35 40 45 Ala Gly Lys Thr Arg AsnIle Ile Glu Lys Met Glu Glu Thr Gly Lys 50 55 60 Asn Cys Val Ile Phe TyrGly Ser Gln Thr Gly Thr Ala Glu Asp Tyr 65 70 75 80 Ala Ser Arg Leu AlaLys Glu Gly Ser Gln Arg Phe Gly Leu Lys Thr 85 90 95 Met Val Ala Asp LeuGlu Glu Tyr Asp Tyr Glu Asn Leu Asp Gln Phe 100 105 110 Pro Glu Asp LysVal Ala Phe Phe Val Leu Ala Thr Tyr Gly Glu Gly 115 120 125 Glu Pro ThrAsp Asn Ala Val Glu Phe Tyr Gln Phe Phe Thr Gly Asp 130 135 140 Asp ValAla Phe Glu Ser Ala Ser Ala Asp Glu Lys Pro Leu Ser Lys 145 150 155 160Leu Lys Tyr Val Ala Phe Gly Leu Gly Asn Asn Thr Tyr Glu His Tyr 165 170175 Asn Ala Met Val Arg Gln Val Asp Ala Ala Phe Gln Lys Leu Gly Pro 180185 190 Gln Arg Ile Gly Ser Ala Gly Glu Gly Asp Asp Gly Ala Gly Thr Met195 200 205 Glu Glu Asp Phe Leu Ala Trp Lys Glu Pro Met Trp Ala Ala LeuSer 210 215 220 Glu Ser Met Asp Leu Glu Glu Arg Glu Ala Val Tyr Glu ProVal Phe 225 230 235 240 Cys Val Thr Glu Asn Glu Ser Leu Ser Pro Glu AspGlu Thr Val Tyr 245 250 255 Leu Gly Glu Pro Thr Gln Ser His Leu Gln GlyThr Pro Lys Gly Pro 260 265 270 Tyr Ser Ala His Asn Pro Phe Ile Ala ProIle Ala Glu Ser Arg Glu 275 280 285 Leu Phe Thr Val Lys Asp Arg Asn CysLeu His Met Glu Ile Ser Ile 290 295 300 Ala Gly Ser Asn Leu Ser Tyr GlnThr Gly Asp His Ile Ala Val Trp 305 310 315 320 Pro Thr Asn Ala Gly AlaGlu Val Asp Arg Phe Leu Gln Val Phe Gly 325 330 335 Leu Glu Gly Lys ArgAsp Ser Val Ile Asn Ile Lys Gly Ile Asp Val 340 345 350 Thr Ala Lys ValPro Ile Pro Thr Pro Thr Thr Tyr Asp Ala Ala Val 355 360 365 Arg Tyr TyrMet Glu Val Cys Ala Pro Val Ser Arg Gln Phe Val Ala 370 375 380 Thr LeuAla Ala Phe Ala Pro Met Arg Lys Ala Arg Gln Arg Leu Cys 385 390 395 400Val Trp Val Ala Gln Gly Leu Phe Pro Arg Glu Gly His Gln Pro Met 405 410415 Leu Gln His Ala Gln Ala Leu Gln Ser Ile Thr Ser Lys Pro Phe Ser 420425 430 Ala Val Pro Phe Ser Leu Leu Ile Glu Gly Ile Thr Lys Leu Gln Pro435 440 445 Arg Tyr Tyr Ser Ile Ser Ser Ser Ser Leu Val Gln Lys Asp LysIle 450 455 460 Ser Ile Thr Ala Val Val Glu Ser Val Arg Leu Pro Gly AlaSer His 465 470 475 480 Met Val Lys Gly Val Thr Thr Asn Tyr Leu Leu AlaLeu Lys Gln Lys 485 490 495 Gln Asn Gly Arg Ser Leu Ser Arg Pro Ser ArgLeu Asp Leu Leu His 500 505 510 His Gly Pro Arg Asn Lys Tyr Asp Gly IleHis Val Pro Val His Val 515 520 525 Arg His Ser Asn Phe Lys Leu Pro SerAsp Pro Ser Arg Pro Ile Ile 530 535 540 Met Val Gly Pro Gly Thr Gly ValAla Pro Phe Arg Gly Phe Ile Gln 545 550 555 560 Glu Arg Ala Ala Leu AlaAla Lys Gly Glu Lys Val Gly Pro Thr Val 565 570 575 Leu Phe Phe Gly CysArg Lys Ser Asp Glu Asp Phe Leu Tyr Lys Asp 580 585 590 Glu Trp Lys ThrTyr Gln Asp Gln Leu Gly Asp Asn Leu Lys Ile Ile 595 600 605 Thr Ala PheSer Arg Glu Gly Pro Gln Lys Val Tyr Val Gln His Arg 610 615 620 Leu ArgGlu His Ser Glu Leu Val Ser Asp Leu Leu Lys Gln Lys Ala 625 630 635 640Thr Phe Tyr Val Cys Gly Asp Ala Ala Asn Met Ala Arg Glu Val Asn 645 650655 Leu Val Leu Gly Gln Ile Ile Ala Ala Gln Arg Gly Leu Pro Ala Glu 660665 670 Lys Gly Glu Glu Met Val Lys His Met Arg Arg Arg Gly Arg Tyr Gln675 680 685 Glu Asp Val Trp Ser 690 39 678 PRT mouse 39 Met Gly Asp SerHis Glu Asp Thr Ser Ala Thr Val Pro Glu Ala Val 1 5 10 15 Ala Glu GluVal Ser Leu Phe Ser Thr Thr Asp Ile Val Leu Phe Ser 20 25 30 Leu Ile ValGly Val Leu Thr Tyr Trp Phe Ile Phe Lys Lys Lys Lys 35 40 45 Glu Glu IlePro Glu Phe Ser Lys Ile Gln Thr Thr Ala Pro Pro Val 50 55 60 Lys Glu SerSer Phe Val Glu Lys Met Lys Lys Thr Gly Arg Asn Ile 65 70 75 80 Ile ValPhe Tyr Gly Ser Gln Thr Gly Thr Ala Glu Glu Phe Ala Asn 85 90 95 Arg LeuSer Lys Asp Ala His Arg Tyr Gly Met Arg Gly Met Ser Ala 100 105 110 AspPro Glu Glu Tyr Asp Leu Ala Asp Leu Ser Ser Leu Pro Glu Ile 115 120 125Asp Lys Ser Leu Val Val Phe Cys Met Ala Thr Tyr Gly Glu Gly Asp 130 135140 Pro Thr Asp Asn Ala Gln Asp Phe Tyr Asp Trp Leu Gln Glu Thr Asp 145150 155 160 Val Asp Leu Thr Gly Val Lys Phe Ala Val Phe Gly Leu Gly AsnLys 165 170 175 Thr Tyr Glu His Phe Asn Ala Met Gly Lys Tyr Val Asp GlnArg Leu 180 185 190 Glu Gln Leu Gly Ala Gln Arg Ile Phe Glu Leu Gly LeuGly Asp Asp 195 200 205 Asp Gly Asn Leu Glu Glu Asp Phe Ile Thr Trp ArgGlu Gln Phe Trp 210 215 220 Pro Ala Val Cys Glu Phe Phe Gly Val Glu AlaThr Gly Glu Glu Ser 225 230 235 240 Ser Ile Arg Gln Tyr Glu Leu Val ValHis Glu Asp Met Asp Thr Ala 245 250 255 Lys Val Tyr Thr Gly Glu Met GlyArg Leu Lys Ser Tyr Glu Asn Gln 260 265 270 Lys Pro Pro Phe Asp Ala LysAsn Pro Phe Leu Ala Ala Val Thr Thr 275 280 285 Asn Arg Lys Leu Asn GlnGly Thr Glu Arg His Leu Met His Leu Glu 290 295 300 Leu Asp Ile Ser AspSer Lys Ile Arg Tyr Glu Ser Gly Asp His Val 305 310 315 320 Ala Val TyrPro Ala Asn Asp Ser Thr Leu Val Asn Gln Ile Gly Glu 325 330 335 Ile LeuGly Ala Asp Leu Asp Val Ile Met Ser Leu Asn Asn Leu Asp 340 345 350 GluGlu Ser Asn Lys Lys His Pro Phe Pro Cys Pro Thr Thr Tyr Arg 355 360 365Thr Ala Leu Thr Tyr Tyr Leu Asp Ile Thr Asn Pro Pro Arg Thr Asn 370 375380 Val Leu Tyr Glu Leu Ala Gln Tyr Ala Ser Glu Pro Ser Glu Gln Glu 385390 395 400 His Leu His Lys Met Ala Ser Ser Ser Gly Glu Gly Lys Glu LeuTyr 405 410 415 Leu Ser Trp Val Val Glu Ala Arg Arg His Ile Leu Ala IleLeu Gln 420 425 430 Asp Tyr Pro Ser Leu Arg Pro Pro Ile Asp His Leu CysGlu Leu Leu 435 440 445 Pro Arg Leu Gln Ala Arg Tyr Tyr Ser Ile Ala SerSer Ser Lys Val 450 455 460 His Pro Asn Ser Val His Ile Cys Ala Val AlaVal Glu Tyr Glu Ala 465 470 475 480 Lys Ser Gly Arg Val Asn Lys Gly ValAla Thr Ser Trp Leu Arg Thr 485 490 495 Lys Glu Pro Ala Gly Glu Asn GlyArg Arg Ala Leu Val Pro Met Phe 500 505 510 Val Arg Lys Ser Gln Phe ArgLeu Pro Phe Lys Pro Thr Thr Pro Val 515 520 525 Ile Met Val Gly Pro GlyThr Gly Val Ala Pro Phe Met Gly Phe Ile 530 535 540 Gln Glu Arg Ala TrpLeu Arg Glu Gln Gly Lys Glu Val Gly Glu Thr 545 550 555 560 Leu Leu TyrTyr Gly Cys Arg Arg Ser Asp Glu Asp Tyr Leu Tyr Arg 565 570 575 Glu GluLeu Ala Arg Phe His Lys Asp Gly Ala Leu Thr Gln Leu Asn 580 585 590 ValAla Phe Ser Arg Glu Gln Ala His Lys Val Tyr Val Gln His Leu 595 600 605Leu Lys Arg Asp Lys Glu His Leu Trp Lys Leu Ile His Glu Gly Gly 610 615620 Ala His Ile Tyr Val Cys Gly Asp Ala Arg Asn Met Ala Lys Asp Val 625630 635 640 Gln Asn Thr Phe Tyr Asp Ile Val Ala Glu Phe Gly Pro Met GluHis 645 650 655 Thr Gln Ala Val Asp Tyr Val Lys Lys Leu Met Thr Lys GlyArg Tyr 660 665 670 Ser Leu Asp Val Trp Ser 675 40 17 DNA baceriophageM13 reverse primer 40 caggaaacag ctatgac 17 41 20 DNA bacteriophage T7promoter primer 41 taatacgact cactataggg 20 42 30 DNA Aspergillusochraceus Primer 11alphaOH-for 42 gatcgaattc atgcccttct tcactgggct 30 4337 DNA Aspergillus ochraceus Primer 11alphaOH-rev 43 gatctctagattacacagtt aaactcgcca tatcgat 37 44 20 DNA pFastBacI Primer Bacfwd 44ctgttttcgt aacagttttg 20 45 19 DNA pFastBacI Primer PolyA 45 cctctacaaatgtggtatg 19 46 17 DNA Aspergillus ochraceus Primer 45624-for1 46gagatcaaga ttgcctt 17 47 15 DNA Aspergillus ochraceus Primer 45624-for247 cttcgacgct ctcaa 15 48 17 DNA Aspergillus ochraceus Primer 45624-rev148 gcaatcttga tctcgtt 17 49 2403 DNA human oxidoreductase partial S9046949 ggagactccc acgtggacac cagctccacc gtgtccgagg cggtggccga agaagtatct 60cttttcagca tgacggacat gattctgttt tcgctcatcg tgggtctcct aacctactgg 120ttcctcttca gaaagaaaaa agaagaagtc cccgagttca ccaaaattca gacattgacc 180tcctctgtca gagagagcag ctttgtggaa aagatgaaga aaacggggag gaacatcatc 240gtgttctacg gctcccagac ggggactgca gaggagtttg ccaaccgcct gtccaaggac 300gcccaccgct acgggatgcg aggcatgtca gcggaccctg aggagtatga cctggccgac 360ctgagcagcc tgccagagat cgacaacgcc ctggtggttt tctgcatggc cacctacggt 420gagggagacc ccaccgacaa tgcccaggac ttctacgact ggctgcagga gacagacgtg 480gatctctctg gggtcaagtt cgcggtgttt ggtcttggga acaagaccta cgagcacttc 540aatgccatgg gcaagtacgt ggacaagcgg ctggagcagc tcggcgccca gcgcatcttt 600gagctggggt tgggcgacga cgatgggaac ttggaggagg acttcatcac ctggcgagag 660cagttctggc cggccgtgtg tgaacacttt ggggtggaag ccactggcga ggagtccagc 720attcgccagt acgagcttgt ggtccacacc gacatagatg cggccaaggt gtacatgggg 780gagatgggcc ggctgaagag ctacgagaac cagaagcccc cctttgatgc caagaatccg 840ttcctggctg cagtcaccac caaccggaag ctgaaccagg gaaccgagcg ccacctcatg 900cacctggaat tggacatctc ggactccaaa atcaggtatg aatctgggga ccacgtggct 960gtgtacccag ccaacgactc tgctctcgtc aaccagctgg gcaaaatcct gggtgccgac 020ctggacgtcg tcatgtccct gaacaacctg gatgaggagt ccaacaagaa gcacccattc 080ccgtgcccta cgtcctaccg cacggccctc acctactacc tggacatcac caacccgccg 140cgtaccaacg tgctgtacga gctggcgcag tacgcctcgg agccctcgga gcaggagctg 200ctgcgcaaga tggcctcctc ctccggcgag ggcaaggagc tgtacctgag ctgggtggtg 260gaggcccgga ggcacatcct ggccatcctg caggactgcc cgtccctgcg gccccccatc 320gaccacctgt gtgagctgct gccgcgcctg caggcccgct actactccat cgcctcatcc 380tccaaggtcc accccaactc tgtgcacatc tgtgcggtgg ttgtggagta cgagaccaag 440gccggccgca tcaacaaggg cgtggccacc aactggctgc gggccaagga gcctgtcggg 500gagaacggcg gccgtgcgct ggtgcccatg ttcgtgcgca agtcccagtt acgcctgccc 560ttcaaggcca ccacgcctgt catcatggtg ggccccggca ccgggtggca ccctttcata 620ggcttcatcc aggagcgggc ctggctgcga cagcagggca aggaggtggg ggagacgctg 680ctgtactacg gctgccgccg ctcggatgag gactacctgt accgggagga gctggcgcag 740ttccacaggg acggtgcgct cacccagctc aacgtggcct tctcccggga gcagtcccac 800aaggtctacg tccagcacct gctaaagcaa gaccgagagc acctgtggaa gttgatcgaa 860ggcggtgccc acatctacgt ctgtggggat gcacggaaca tggccaggga tgtgcagaac 920accttctacg acatcgtggc tgagctcggg gccatggagc acgcgcaggc ggtggactac 980atcaagaaac tgatgaccaa gggccgctac tccctggacg tgtggagcta ggggcctgcc 040tgccccaccc accccacaga ctccggcctg taatcagctc tcctggctcc ctcccgtagt 100ctcctgggtg tgtttggctt ggccttggca tgggcgcagg cccagtgaca aagactcctc 160tgggcctggg gtgcatcctc ctcagccccc aggccaggtg aggtccaccg gcccctggca 220gcacagccca gggcctgcat gggggcaccg ggctccatgc ctctggagcc tctggccctc 280ggtggctgca cagaagggct ctttctctct gctgagctgg cccagcccct ccacgtgatt 340tccagtgagt gtaaataatt ttaaataacc tctggccctt ggaataaagt tctgttttct 400gta 403 50 676 PRT human cytochrome P450 reductase, partial AAB21814 50Gly Asp Ser His Val Asp Thr Ser Ser Thr Val Ser Glu Ala Val Ala 1 5 1015 Glu Glu Val Ser Leu Phe Ser Met Thr Asp Met Ile Leu Phe Ser Leu 20 2530 Ile Val Gly Leu Leu Thr Tyr Trp Phe Leu Phe Arg Lys Lys Lys Glu 35 4045 Glu Val Pro Glu Phe Thr Lys Ile Gln Thr Leu Thr Ser Ser Val Arg 50 5560 Glu Ser Ser Phe Val Glu Lys Met Lys Lys Thr Gly Arg Asn Ile Ile 65 7075 80 Val Phe Tyr Gly Ser Gln Thr Gly Thr Ala Glu Glu Phe Ala Asn Arg 8590 95 Leu Ser Lys Asp Ala His Arg Tyr Gly Met Arg Gly Met Ser Ala Asp100 105 110 Pro Glu Glu Tyr Asp Leu Ala Asp Leu Ser Ser Leu Pro Glu IleAsp 115 120 125 Asn Ala Leu Val Val Phe Cys Met Ala Thr Tyr Gly Glu GlyAsp Pro 130 135 140 Thr Asp Asn Ala Gln Asp Phe Tyr Asp Trp Leu Gln GluThr Asp Val 145 150 155 160 Asp Leu Ser Gly Val Lys Phe Ala Val Phe GlyLeu Gly Asn Lys Thr 165 170 175 Tyr Glu His Phe Asn Ala Met Gly Lys TyrVal Asp Lys Arg Leu Glu 180 185 190 Gln Leu Gly Ala Gln Arg Ile Phe GluLeu Gly Leu Gly Asp Asp Asp 195 200 205 Gly Asn Leu Glu Glu Asp Phe IleThr Trp Arg Glu Gln Phe Trp Pro 210 215 220 Ala Val Cys Glu His Phe GlyVal Glu Ala Thr Gly Glu Glu Ser Ser 225 230 235 240 Ile Arg Gln Tyr GluLeu Val Val His Thr Asp Ile Asp Ala Ala Lys 245 250 255 Val Tyr Met GlyGlu Met Gly Arg Leu Lys Ser Tyr Glu Asn Gln Lys 260 265 270 Pro Pro PheAsp Ala Lys Asn Pro Phe Leu Ala Ala Val Thr Thr Asn 275 280 285 Arg LysLeu Asn Gln Gly Thr Glu Arg His Leu Met His Leu Glu Leu 290 295 300 AspIle Ser Asp Ser Lys Ile Arg Tyr Glu Ser Gly Asp His Val Ala 305 310 315320 Val Tyr Pro Ala Asn Asp Ser Ala Leu Val Asn Gln Leu Gly Lys Ile 325330 335 Leu Gly Ala Asp Leu Asp Val Val Met Ser Leu Asn Asn Leu Asp Glu340 345 350 Glu Ser Asn Lys Lys His Pro Phe Pro Cys Pro Thr Ser Tyr ArgThr 355 360 365 Ala Leu Thr Tyr Tyr Leu Asp Ile Thr Asn Pro Pro Arg ThrAsn Val 370 375 380 Leu Tyr Glu Leu Ala Gln Tyr Ala Ser Glu Pro Ser GluGln Glu Leu 385 390 395 400 Leu Arg Lys Met Ala Ser Ser Ser Gly Glu GlyLys Glu Leu Tyr Leu 405 410 415 Ser Trp Val Val Glu Ala Arg Arg His IleLeu Ala Ile Leu Gln Asp 420 425 430 Cys Pro Ser Leu Arg Pro Pro Ile AspHis Leu Cys Glu Leu Leu Pro 435 440 445 Arg Leu Gln Ala Arg Tyr Tyr SerIle Ala Ser Ser Ser Lys Val His 450 455 460 Pro Asn Ser Val His Ile CysAla Val Val Val Glu Tyr Glu Thr Lys 465 470 475 480 Ala Gly Arg Ile AsnLys Gly Val Ala Thr Asn Trp Leu Arg Ala Lys 485 490 495 Glu Pro Val GlyGlu Asn Gly Gly Arg Ala Leu Val Pro Met Phe Val 500 505 510 Arg Lys SerGln Leu Arg Leu Pro Phe Lys Ala Thr Thr Pro Val Ile 515 520 525 Met ValGly Pro Gly Thr Gly Trp His Pro Phe Ile Gly Phe Ile Gln 530 535 540 GluArg Ala Trp Leu Arg Gln Gln Gly Lys Glu Val Gly Glu Thr Leu 545 550 555560 Leu Tyr Tyr Gly Cys Arg Arg Ser Asp Glu Asp Tyr Leu Tyr Arg Glu 565570 575 Glu Leu Ala Gln Phe His Arg Asp Gly Ala Leu Thr Gln Leu Asn Val580 585 590 Ala Phe Ser Arg Glu Gln Ser His Lys Val Tyr Val Gln His LeuLeu 595 600 605 Lys Gln Asp Arg Glu His Leu Trp Lys Leu Ile Glu Gly GlyAla His 610 615 620 Ile Tyr Val Cys Gly Asp Ala Arg Asn Met Ala Arg AspVal Gln Asn 625 630 635 640 Thr Phe Tyr Asp Ile Val Ala Glu Leu Gly AlaMet Glu His Ala Gln 645 650 655 Ala Val Asp Tyr Ile Lys Lys Leu Met ThrLys Gly Arg Tyr Ser Leu 660 665 670 Asp Val Trp Ser 675 51 677 PRT humanNADPH-ferrihemoprotein reductase A60557 51 Met Gly Asp Ser His Val AspThr Ser Ser Thr Val Ser Glu Ala Val 1 5 10 15 Ala Glu Glu Val Ser LeuPhe Ser Met Thr Asp Met Ile Leu Phe Ser 20 25 30 Leu Ile Val Gly Leu LeuThr Tyr Trp Phe Leu Phe Arg Lys Lys Lys 35 40 45 Glu Glu Val Pro Glu PheThr Lys Ile Gln Thr Leu Thr Ser Ser Val 50 55 60 Arg Glu Ser Ser Phe ValGlu Lys Met Lys Lys Thr Gly Arg Asn Ile 65 70 75 80 Ile Val Phe Tyr GlySer Gln Thr Gly Thr Ala Glu Glu Phe Ala Asn 85 90 95 Arg Leu Ser Lys AspAla His Arg Tyr Gly Met Arg Gly Met Ser Ala 100 105 110 Asp Pro Glu GluTyr Asp Leu Ala Asp Leu Ser Ser Leu Pro Glu Ile 115 120 125 Asp Asn AlaLeu Val Val Phe Cys Met Ala Thr Tyr Gly Glu Gly Asp 130 135 140 Pro ThrAsp Asn Ala Gln Asp Phe Tyr Asp Trp Leu Gln Glu Thr Asp 145 150 155 160Val Asp Leu Ser Gly Val Lys Phe Ala Val Phe Gly Leu Gly Asn Lys 165 170175 Thr Tyr Glu His Phe Asn Ala Met Gly Lys Tyr Val Asp Lys Arg Leu 180185 190 Glu Gln Leu Gly Ala Gln Arg Ile Phe Glu Leu Gly Leu Gly Asp Asp195 200 205 Asp Gly Asn Leu Glu Glu Asp Phe Ile Thr Trp Arg Glu Gln PheTrp 210 215 220 Pro Ala Val Cys Glu His Phe Gly Val Glu Ala Thr Gly GluGlu Ser 225 230 235 240 Ser Ile Arg Gln Tyr Glu Leu Val Val His Thr AspIle Asp Ala Ala 245 250 255 Lys Val Tyr Met Gly Glu Met Gly Arg Leu LysSer Tyr Glu Asn Gln 260 265 270 Lys Pro Pro Phe Asp Ala Lys Asn Pro PheLeu Ala Ala Val Thr Thr 275 280 285 Asn Arg Lys Leu Asn Gln Gly Thr GluArg His Leu Met His Leu Glu 290 295 300 Leu Asp Ile Ser Asp Ser Lys IleArg Tyr Glu Ser Gly Asp His Val 305 310 315 320 Ala Val Tyr Pro Ala AsnAsp Ser Ala Leu Val Asn Gln Leu Gly Lys 325 330 335 Ile Leu Gly Ala AspLeu Asp Val Val Met Ser Leu Asn Asn Leu Asp 340 345 350 Glu Glu Ser AsnLys Lys His Pro Phe Pro Cys Pro Thr Ser Tyr Arg 355 360 365 Thr Ala LeuThr Tyr Tyr Leu Asp Ile Thr Asn Pro Pro Arg Thr Asn 370 375 380 Val LeuTyr Glu Leu Ala Gln Tyr Ala Ser Glu Pro Ser Glu Gln Glu 385 390 395 400Leu Leu Arg Lys Met Ala Ser Ser Ser Gly Glu Gly Lys Glu Leu Tyr 405 410415 Leu Ser Trp Val Val Glu Ala Arg Arg His Ile Leu Ala Ile Leu Gln 420425 430 Asp Cys Pro Ser Leu Arg Pro Pro Ile Asp His Leu Cys Glu Leu Leu435 440 445 Pro Arg Leu Gln Ala Arg Tyr Tyr Ser Ile Ala Ser Ser Ser LysVal 450 455 460 His Pro Asn Ser Val His Ile Cys Ala Val Val Val Glu TyrGlu Thr 465 470 475 480 Lys Ala Gly Arg Ile Asn Lys Gly Val Ala Thr AsnTrp Leu Arg Ala 485 490 495 Lys Glu Pro Ala Gly Glu Asn Gly Gly Arg AlaLeu Val Pro Met Phe 500 505 510 Val Arg Lys Ser Gln Phe Arg Leu Pro PheLys Ala Thr Thr Pro Val 515 520 525 Ile Met Val Gly Pro Gly Thr Gly ValAla Pro Phe Ile Gly Phe Ile 530 535 540 Gln Glu Arg Ala Trp Leu Arg GlnGln Gly Lys Glu Val Gly Glu Thr 545 550 555 560 Leu Leu Tyr Tyr Gly CysArg Arg Ser Asp Glu Asp Tyr Leu Tyr Arg 565 570 575 Glu Glu Leu Ala GlnPhe His Arg Asp Gly Ala Leu Thr Gln Leu Asn 580 585 590 Val Ala Phe SerArg Glu Gln Ser His Lys Val Tyr Val Gln His Leu 595 600 605 Leu Lys GlnAsp Arg Glu His Leu Trp Lys Leu Ile Glu Gly Gly Ala 610 615 620 His IleTyr Val Cys Gly Asp Ala Arg Asn Met Ala Arg Asp Val Gln 625 630 635 640Asn Thr Phe Tyr Asp Ile Val Ala Glu Leu Gly Ala Met Glu His Ala 645 650655 Gln Ala Val Asp Tyr Ile Lys Lys Leu Met Thr Lys Gly Arg Tyr Ser 660665 670 Leu Asp Val Trp Ser 675 52 677 PRT human NADPH-CYTOCHROME P450REDUCTASE P16435 52 Met Gly Asp Ser His Val Asp Thr Ser Ser Thr Val SerGlu Ala Val 1 5 10 15 Ala Glu Glu Val Ser Leu Phe Ser Met Thr Asp MetIle Leu Phe Ser 20 25 30 Leu Ile Val Gly Leu Leu Thr Tyr Trp Phe Leu PheArg Lys Lys Lys 35 40 45 Glu Glu Val Pro Glu Phe Thr Lys Ile Gln Thr LeuThr Ser Ser Val 50 55 60 Arg Glu Ser Ser Phe Val Glu Lys Met Lys Lys ThrGly Arg Asn Ile 65 70 75 80 Ile Val Phe Tyr Gly Ser Gln Thr Gly Thr AlaGlu Glu Phe Ala Asn 85 90 95 Arg Leu Ser Lys Asp Ala His Arg Tyr Gly MetArg Gly Met Ser Ala 100 105 110 Asp Pro Glu Glu Tyr Asp Leu Ala Asp LeuSer Ser Leu Pro Glu Ile 115 120 125 Asp Asn Ala Leu Val Val Phe Cys MetAla Thr Tyr Gly Glu Gly Asp 130 135 140 Pro Thr Asp Asn Ala Gln Asp PheTyr Asp Trp Leu Gln Glu Thr Asp 145 150 155 160 Val Asp Leu Ser Gly ValLys Phe Ala Val Phe Gly Leu Gly Asn Lys 165 170 175 Thr Tyr Glu His PheAsn Ala Met Gly Lys Tyr Val Asp Lys Arg Leu 180 185 190 Glu Gln Leu GlyAla Gln Arg Ile Phe Glu Leu Gly Leu Gly Asp Asp 195 200 205 Asp Gly AsnLeu Glu Glu Asp Phe Ile Thr Trp Arg Glu Gln Phe Trp 210 215 220 Pro AlaVal Cys Glu His Phe Gly Val Glu Ala Thr Gly Glu Glu Ser 225 230 235 240Ser Ile Arg Gln Tyr Glu Leu Val Val His Thr Asp Ile Asp Ala Ala 245 250255 Lys Val Tyr Met Gly Glu Met Gly Arg Leu Lys Ser Tyr Glu Asn Gln 260265 270 Lys Pro Pro Phe Asp Ala Lys Asn Pro Phe Leu Ala Ala Val Thr Thr275 280 285 Asn Arg Lys Leu Asn Gln Gly Thr Glu Arg His Leu Met His LeuGlu 290 295 300 Leu Asp Ile Ser Asp Ser Lys Ile Arg Tyr Glu Ser Gly AspHis Val 305 310 315 320 Ala Val Tyr Pro Ala Asn Asp Ser Ala Leu Val AsnGln Leu Gly Lys 325 330 335 Ile Leu Gly Ala Asp Leu Asp Val Val Met SerLeu Asn Asn Leu Asp 340 345 350 Glu Glu Ser Asn Lys Lys His Pro Phe ProCys Pro Thr Ser Tyr Arg 355 360 365 Thr Ala Leu Thr Tyr Tyr Leu Asp IleThr Asn Pro Pro Arg Thr Asn 370 375 380 Val Leu Tyr Glu Leu Ala Gln TyrAla Ser Glu Pro Ser Glu Gln Glu 385 390 395 400 Leu Leu Arg Lys Met AlaSer Ser Ser Gly Glu Gly Lys Glu Leu Tyr 405 410 415 Leu Ser Trp Val ValGlu Ala Arg Arg His Ile Leu Ala Ile Leu Gln 420 425 430 Asp Cys Pro SerLeu Arg Pro Pro Ile Asp His Leu Cys Glu Leu Leu 435 440 445 Pro Arg LeuGln Ala Arg Tyr Tyr Ser Ile Ala Ser Ser Ser Lys Val 450 455 460 His ProAsn Ser Val His Ile Cys Ala Val Val Val Glu Tyr Glu Thr 465 470 475 480Lys Ala Gly Arg Ile Asn Lys Gly Val Ala Thr Asn Trp Leu Arg Ala 485 490495 Lys Glu Pro Ala Gly Glu Asn Gly Gly Arg Ala Leu Val Pro Met Phe 500505 510 Val Arg Lys Ser Gln Phe Arg Leu Pro Phe Lys Ala Thr Thr Pro Val515 520 525 Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Ile Gly PheIle 530 535 540 Gln Glu Arg Ala Trp Leu Arg Gln Gln Gly Lys Glu Val GlyGlu Thr 545 550 555 560 Leu Leu Tyr Tyr Gly Cys Arg Arg Ser Asp Glu AspTyr Leu Tyr Arg 565 570 575 Glu Glu Leu Ala Gln Phe His Arg Asp Gly AlaLeu Thr Gln Leu Asn 580 585 590 Val Ala Phe Ser Arg Glu Gln Ser His LysVal Tyr Val Gln His Leu 595 600 605 Leu Lys Gln Asp Arg Glu His Leu TrpLys Leu Ile Glu Gly Gly Ala 610 615 620 His Ile Tyr Val Cys Gly Asp AlaArg Asn Met Ala Arg Asp Val Gln 625 630 635 640 Asn Thr Phe Tyr Asp IleVal Ala Glu Leu Gly Ala Met Glu His Ala 645 650 655 Gln Ala Val Asp TyrIle Lys Lys Leu Met Thr Lys Gly Arg Tyr Ser 660 665 670 Leu Asp Val TrpSer 675 53 679 PRT Rabbit NADPH-CYTOCHROME P450 REDUCTASE P00389 53 MetAla Asp Ser His Gly Asp Thr Gly Ala Thr Met Pro Glu Ala Ala 1 5 10 15Ala Gln Glu Ala Ser Val Phe Ser Met Thr Asp Val Val Leu Phe Ser 20 25 30Leu Ile Val Gly Leu Ile Thr Tyr Trp Phe Leu Phe Arg Lys Lys Lys 35 40 45Glu Glu Val Pro Glu Phe Thr Lys Ile Gln Ala Pro Thr Ser Ser Ser 50 55 60Val Lys Glu Ser Ser Phe Val Glu Lys Met Lys Lys Thr Gly Arg Asn 65 70 7580 Ile Val Val Phe Tyr Gly Ser Gln Thr Gly Thr Ala Glu Glu Phe Ala 85 9095 Asn Arg Leu Ser Lys Asp Ala His Arg Tyr Gly Met Arg Gly Met Ala 100105 110 Ala Asp Pro Glu Glu Tyr Asp Leu Ala Asp Leu Ser Ser Leu Pro Glu115 120 125 Ile Asn Asn Ala Leu Ala Val Phe Cys Met Ala Thr Tyr Gly GluGly 130 135 140 Asp Pro Thr Asp Asn Ala Gln Asp Phe Tyr Asp Trp Leu GlnGlu Thr 145 150 155 160 Asp Val Asp Leu Ser Gly Val Lys Tyr Ala Val PheGly Leu Gly Asn 165 170 175 Lys Thr Tyr Glu His Phe Asn Ala Met Gly LysTyr Val Asp Gln Arg 180 185 190 Leu Glu Gln Leu Gly Ala Gln Arg Ile PheGlu Leu Gly Met Gly Asp 195 200 205 Asp Asp Ala Asn Leu Glu Glu Asp PheIle Thr Trp Arg Glu Gln Phe 210 215 220 Trp Pro Ala Val Cys Glu His PheGly Val Glu Ala Thr Gly Glu Glu 225 230 235 240 Ser Ser Ile Arg Gln TyrGlu Leu Val Leu His Thr Asp Ile Asp Val 245 250 255 Ala Lys Val Tyr GlnGly Glu Met Gly Arg Leu Lys Ser Tyr Glu Asn 260 265 270 Gln Lys Pro ProPhe Asp Ala Lys Asn Pro Phe Leu Ala Thr Val Thr 275 280 285 Thr Asn ArgLys Leu Asn Gln Gly Thr Glu Arg His Leu Met His Leu 290 295 300 Glu LeuAsp Ile Ser Asp Ser Lys Ile Arg Tyr Glu Ser Gly Asp His 305 310 315 320Val Ala Val Tyr Pro Ala Asn Asp Ser Ala Leu Val Asn Gln Leu Gly 325 330335 Glu Ile Leu Gly Ala Asp Leu Asp Val Val Met Ser Leu Asn Asn Leu 340345 350 Asp Glu Glu Ser Asn Lys Lys His Pro Phe Pro Cys Pro Thr Ser Tyr355 360 365 Arg Thr Ala Leu Thr Tyr Tyr Leu Asp Ile Thr Asn Pro Pro ArgThr 370 375 380 Asn Val Leu Tyr Glu Leu Ala Gln Tyr Ala Ala Asp Pro AlaGlu Gln 385 390 395 400 Glu Gln Leu Arg Lys Met Ala Ser Ser Ser Gly GluGly Lys Glu Leu 405 410 415 Tyr Leu Ser Trp Val Val Glu Ala Arg Arg HisIle Leu Ala Ile Leu 420 425 430 Gln Asp Tyr Pro Ser Leu Arg Pro Pro IleAsp His Leu Cys Glu Leu 435 440 445 Leu Pro Arg Leu Gln Ala Arg Tyr TyrSer Ile Ala Ser Ser Ser Lys 450 455 460 Val His Pro Asn Ser Val His IleCys Ala Val Ala Val Glu Tyr Glu 465 470 475 480 Thr Lys Ala Gly Arg LeuAsn Lys Gly Val Ala Thr Ser Trp Leu Arg 485 490 495 Ala Lys Glu Pro AlaGly Glu Asn Gly Gly Arg Ala Leu Val Pro Met 500 505 510 Phe Val Arg LysSer Gln Phe Arg Leu Pro Phe Lys Ala Thr Thr Pro 515 520 525 Val Ile MetVal Gly Pro Gly Thr Gly Val Ala Pro Phe Ile Gly Phe 530 535 540 Ile GlnGlu Arg Ala Trp Leu Arg Gln Gln Gly Lys Glu Val Gly Glu 545 550 555 560Thr Leu Leu Tyr Tyr Gly Cys Arg Arg Ala Ala Glu Asp Tyr Leu Tyr 565 570575 Arg Glu Glu Leu Ala Gly Phe Gln Lys Asp Gly Thr Leu Ser Gln Leu 580585 590 Asn Val Ala Phe Ser Arg Glu Gln Ala Gln Lys Val Tyr Val Gln His595 600 605 Leu Leu Arg Arg Asp Lys Glu His Leu Trp Arg Leu Ile His GluGly 610 615 620 Gly Ala His Ile Tyr Val Cys Gly Asp Ala Arg Asn Met AlaArg Asp 625 630 635 640 Val Gln Asn Thr Phe Tyr Asp Ile Val Ala Glu LeuGly Ala Met Glu 645 650 655 His Ala Gln Ala Val Asp Tyr Val Lys Lys LeuMet Thr Lys Gly Arg 660 665 670 Tyr Ser Leu Asp Val Trp Ser 675 54 678PRT Rat NADPH-CYTOCHROME P450 REDUCTASE P00388 54 Met Gly Asp Ser HisGlu Asp Thr Ser Ala Thr Met Pro Glu Ala Val 1 5 10 15 Ala Glu Glu ValSer Leu Phe Ser Thr Thr Asp Met Val Leu Phe Ser 20 25 30 Leu Ile Val GlyVal Leu Thr Tyr Trp Phe Ile Phe Arg Lys Lys Lys 35 40 45 Glu Glu Ile ProGlu Phe Ser Lys Ile Gln Thr Thr Ala Pro Pro Val 50 55 60 Lys Glu Ser SerPhe Val Glu Lys Met Lys Lys Thr Gly Arg Asn Ile 65 70 75 80 Ile Val PheTyr Gly Ser Gln Thr Gly Thr Ala Glu Glu Phe Ala Asn 85 90 95 Arg Leu SerLys Asp Ala His Arg Tyr Gly Met Arg Gly Met Ser Ala 100 105 110 Asp ProGlu Glu Tyr Asp Leu Ala Asp Leu Ser Ser Leu Pro Glu Ile 115 120 125 AspLys Ser Leu Val Val Phe Cys Met Ala Thr Tyr Gly Glu Gly Asp 130 135 140Pro Thr Asp Asn Ala Gln Asp Phe Tyr Asp Trp Leu Gln Glu Thr Asp 145 150155 160 Val Asp Leu Thr Gly Val Lys Phe Ala Val Phe Gly Leu Gly Asn Lys165 170 175 Thr Tyr Glu His Phe Asn Ala Met Gly Lys Tyr Val Asp Gln ArgLeu 180 185 190 Glu Gln Leu Gly Ala Gln Arg Ile Phe Glu Leu Gly Leu GlyAsp Asp 195 200 205 Asp Gly Asn Leu Glu Glu Asp Phe Ile Thr Trp Arg GluGln Phe Trp 210 215 220 Pro Ala Val Cys Glu Phe Phe Gly Val Glu Ala ThrGly Glu Glu Ser 225 230 235 240 Ser Ile Arg Gln Tyr Glu Leu Val Val HisGlu Asp Met Asp Val Ala 245 250 255 Lys Val Tyr Thr Gly Glu Met Gly ArgLeu Lys Ser Tyr Glu Asn Gln 260 265 270 Lys Pro Pro Phe Asp Ala Lys AsnPro Phe Leu Ala Ala Val Thr Ala 275 280 285 Asn Arg Lys Leu Asn Gln GlyThr Glu Arg His Leu Met His Leu Glu 290 295 300 Leu Asp Ile Ser Asp SerLys Ile Arg Tyr Glu Ser Gly Asp His Val 305 310 315 320 Ala Val Tyr ProAla Asn Asp Ser Ala Leu Val Asn Gln Ile Gly Glu 325 330 335 Ile Leu GlyAla Asp Leu Asp Val Ile Met Ser Leu Asn Asn Leu Asp 340 345 350 Glu GluSer Asn Lys Lys His Pro Phe Pro Cys Pro Thr Thr Tyr Arg 355 360 365 ThrAla Leu Thr Tyr Tyr Leu Asp Ile Thr Asn Pro Pro Arg Thr Asn 370 375 380Val Leu Tyr Glu Leu Ala Gln Tyr Ala Ser Glu Pro Ser Glu Gln Glu 385 390395 400 His Leu His Lys Met Ala Ser Ser Ser Gly Glu Gly Lys Glu Leu Tyr405 410 415 Leu Ser Trp Val Val Glu Ala Arg Arg His Ile Leu Ala Ile LeuGln 420 425 430 Asp Tyr Pro Ser Leu Arg Pro Pro Ile Asp His Leu Cys GluLeu Leu 435 440 445 Pro Arg Leu Gln Ala Arg Tyr Tyr Ser Ile Ala Ser SerSer Lys Val 450 455 460 His Pro Asn Ser Val His Ile Cys Ala Val Ala ValGlu Tyr Glu Ala 465 470 475 480 Lys Ser Gly Arg Val Asn Lys Gly Val AlaThr Ser Trp Leu Arg Ala 485 490 495 Lys Glu Pro Ala Gly Glu Asn Gly GlyArg Ala Leu Val Pro Met Phe 500 505 510 Val Arg Lys Ser Gln Phe Arg LeuPro Phe Lys Ser Thr Thr Pro Val 515 520 525 Ile Met Val Gly Pro Gly ThrGly Ile Ala Pro Phe Met Gly Phe Ile 530 535 540 Gln Glu Arg Ala Trp LeuArg Glu Gln Gly Lys Glu Val Gly Glu Thr 545 550 555 560 Leu Leu Tyr TyrGly Cys Arg Arg Ser Asp Glu Asp Tyr Leu Tyr Arg 565 570 575 Glu Glu LeuAla Arg Phe His Lys Asp Gly Ala Leu Thr Gln Leu Asn 580 585 590 Val AlaPhe Ser Arg Glu Gln Ala His Lys Val Tyr Val Gln His Leu 595 600 605 LeuLys Arg Asp Arg Glu His Leu Trp Lys Leu Ile His Glu Gly Gly 610 615 620Ala His Ile Tyr Val Cys Gly Asp Ala Arg Asn Met Ala Lys Asp Val 625 630635 640 Gln Asn Thr Phe Tyr Asp Ile Val Ala Glu Phe Gly Pro Met Glu His645 650 655 Thr Gln Ala Val Asp Tyr Val Lys Lys Leu Met Thr Lys Gly ArgTyr 660 665 670 Ser Leu Asp Val Trp Ser 675 55 678 PRT MouseNADPH-CYTOCHROME P450 REDUCTASE P37040 55 Met Gly Asp Ser His Glu AspThr Ser Ala Thr Val Pro Glu Ala Val 1 5 10 15 Ala Glu Glu Val Ser LeuPhe Ser Thr Thr Asp Ile Val Leu Phe Ser 20 25 30 Leu Ile Val Gly Val LeuThr Tyr Trp Phe Ile Phe Lys Lys Lys Lys 35 40 45 Glu Glu Ile Pro Glu PheSer Lys Ile Gln Thr Thr Ala Pro Pro Val 50 55 60 Lys Glu Ser Ser Phe ValGlu Lys Met Lys Lys Thr Gly Arg Asn Ile 65 70 75 80 Ile Val Phe Tyr GlySer Gln Thr Gly Thr Ala Glu Glu Phe Ala Asn 85 90 95 Arg Leu Ser Lys AspAla His Arg Tyr Gly Met Arg Gly Met Ser Ala 100 105 110 Asp Pro Glu GluTyr Asp Leu Ala Asp Leu Ser Ser Leu Pro Glu Ile 115 120 125 Asp Lys SerLeu Val Val Phe Cys Met Ala Thr Tyr Gly Glu Gly Asp 130 135 140 Pro ThrAsp Asn Ala Gln Asp Phe Tyr Asp Trp Leu Gln Glu Thr Asp 145 150 155 160Val Asp Leu Thr Gly Val Lys Phe Ala Val Phe Gly Leu Gly Asn Lys 165 170175 Thr Tyr Glu His Phe Asn Ala Met Gly Lys Tyr Val Asp Gln Arg Leu 180185 190 Glu Gln Leu Gly Ala Gln Arg Ile Phe Glu Leu Gly Leu Gly Asp Asp195 200 205 Asp Gly Asn Leu Glu Glu Asp Phe Ile Thr Trp Arg Glu Gln PheTrp 210 215 220 Pro Ala Val Cys Glu Phe Phe Gly Val Glu Ala Thr Gly GluGlu Ser 225 230 235 240 Ser Ile Arg Gln Tyr Glu Leu Val Val His Glu AspMet Asp Thr Ala 245 250 255 Lys Val Tyr Thr Gly Glu Met Gly Arg Leu LysSer Tyr Glu Asn Gln 260 265 270 Lys Pro Pro Phe Asp Ala Lys Asn Pro PheLeu Ala Ala Val Thr Thr 275 280 285 Asn Arg Lys Leu Asn Gln Gly Thr GluArg His Leu Met His Leu Glu 290 295 300 Leu Asp Ile Ser Asp Ser Lys IleArg Tyr Glu Ser Gly Asp His Val 305 310 315 320 Ala Val Tyr Pro Ala AsnAsp Ser Thr Leu Val Asn Gln Ile Gly Glu 325 330 335 Ile Leu Gly Ala AspLeu Asp Val Ile Met Ser Leu Asn Asn Leu Asp 340 345 350 Glu Glu Ser AsnLys Lys His Pro Phe Pro Cys Pro Thr Thr Tyr Arg 355 360 365 Thr Ala LeuThr Tyr Tyr Leu Asp Ile Thr Asn Pro Pro Arg Thr Asn 370 375 380 Val LeuTyr Glu Leu Ala Gln Tyr Ala Ser Glu Pro Ser Glu Gln Glu 385 390 395 400His Leu His Lys Met Ala Ser Ser Ser Gly Glu Gly Lys Glu Leu Tyr 405 410415 Leu Ser Trp Val Val Glu Ala Arg Arg His Ile Leu Ala Ile Leu Gln 420425 430 Asp Tyr Pro Ser Leu Arg Pro Pro Ile Asp His Leu Cys Glu Leu Leu435 440 445 Pro Arg Leu Gln Ala Arg Tyr Tyr Ser Ile Ala Ser Ser Ser LysVal 450 455 460 His Pro Asn Ser Val His Ile Cys Ala Val Ala Val Glu TyrGlu Ala 465 470 475 480 Lys Ser Gly Arg Val Asn Lys Gly Val Ala Thr SerTrp Leu Arg Thr 485 490 495 Lys Glu Pro Ala Gly Glu Asn Gly Arg Arg AlaLeu Val Pro Met Phe 500 505 510 Val Arg Lys Ser Gln Phe Arg Leu Pro PheLys Pro Thr Thr Pro Val 515 520 525 Ile Met Val Gly Pro Gly Thr Gly ValAla Pro Phe Met Gly Phe Ile 530 535 540 Gln Glu Arg Ala Trp Leu Arg GluGln Gly Lys Glu Val Gly Glu Thr 545 550 555 560 Leu Leu Tyr Tyr Gly CysArg Arg Ser Asp Glu Asp Tyr Leu Tyr Arg 565 570 575 Glu Glu Leu Ala ArgPhe His Lys Asp Gly Ala Leu Thr Gln Leu Asn 580 585 590 Val Ala Phe SerArg Glu Gln Ala His Lys Val Tyr Val Gln His Leu 595 600 605 Leu Lys ArgAsp Lys Glu His Leu Trp Lys Leu Ile His Glu Gly Gly 610 615 620 Ala HisIle Tyr Val Cys Gly Asp Ala Arg Asn Met Ala Lys Asp Val 625 630 635 640Gln Asn Thr Phe Tyr Asp Ile Val Ala Glu Phe Gly Pro Met Glu His 645 650655 Thr Gln Ala Val Asp Tyr Val Lys Lys Leu Met Thr Lys Gly Arg Tyr 660665 670 Ser Leu Asp Val Trp Ser 675 56 678 PRT Pig NADPH-CYTOCHROME P450REDUCTASE P04175 56 Met Gly Asp Ser Asn Val Asp Thr Gly Thr Thr Thr SerGlu Met Val 1 5 10 15 Ala Glu Glu Val Ser Leu Phe Ser Ala Thr Asp MetVal Leu Phe Ser 20 25 30 Leu Ile Val Gly Leu Leu Thr Tyr Trp Phe Ile PheArg Lys Lys Lys 35 40 45 Asp Glu Val Pro Glu Phe Ser Lys Ile Glu Thr ThrThr Ser Ser Val 50 55 60 Lys Asp Ser Ser Phe Val Glu Lys Met Lys Lys ThrGly Arg Asn Ile 65 70 75 80 Ile Val Phe Tyr Gly Ser Gln Thr Gly Thr AlaGlu Glu Phe Ala Asn 85 90 95 Arg Leu Ser Lys Asp Ala His Arg Tyr Gly MetArg Gly Met Ala Ala 100 105 110 Asp Pro Glu Glu Tyr Asp Leu Ser Asp LeuSer Ser Leu Pro Glu Ile 115 120 125 Glu Asn Ala Leu Ala Val Phe Cys MetAla Thr Tyr Gly Glu Gly Asp 130 135 140 Pro Thr Asp Asn Ala Gln Asp PheTyr Asp Trp Leu Gln Glu Ala Asp 145 150 155 160 Val Asp Leu Thr Gly ValLys Tyr Ala Val Phe Gly Leu Gly Asn Lys 165 170 175 Thr Tyr Glu His PheAsn Ala Met Gly Lys Tyr Val Asp Lys Arg Leu 180 185 190 Glu Gln Leu GlyAla Gln Arg Ile Phe Asp Leu Gly Leu Gly Asp Asp 195 200 205 Asp Gly AsnLeu Glu Glu Asp Phe Ile Thr Trp Arg Glu Gln Phe Trp 210 215 220 Pro AlaVal Cys Glu His Phe Gly Val Glu Ala Thr Gly Glu Glu Ser 225 230 235 240Ser Ile Arg Gln Tyr Glu Leu Val Val His Thr Asp Met Asp Thr Ala 245 250255 Val Val Tyr Thr Gly Glu Met Gly Arg Leu Lys Ser Tyr Glu Asn Gln 260265 270 Lys Pro Pro Phe Asp Ala Lys Asn Pro Phe Leu Ala Val Val Thr Thr275 280 285 Asn Arg Lys Leu Asn Gln Gly Thr Glu Arg His Leu Met His LeuGlu 290 295 300 Leu Asp Ile Ser Asp Ser Lys Ile Arg Tyr Glu Ser Gly AspHis Val 305 310 315 320 Ala Val Tyr Pro Ala Asn Asp Ser Ala Leu Val AsnGln Leu Gly Glu 325 330 335 Ile Leu Gly Thr Asp Leu Asp Ile Val Met SerLeu Asn Asn Leu Asp 340 345 350 Glu Glu Ser Asn Lys Arg His Pro Phe ProCys Pro Thr Thr Tyr Arg 355 360 365 Thr Ala Leu Thr Tyr Tyr Leu Asp IleThr Asn Pro Pro Arg Thr Asn 370 375 380 Val Leu Tyr Glu Leu Ala Gln TyrAla Ser Glu Pro Ser Glu Gln Glu 385 390 395 400 Gln Leu Arg Lys Met AlaSer Ser Ser Gly Glu Gly Lys Glu Leu Tyr 405 410 415 Leu Ser Trp Val ValGlu Ala Arg Arg His Ile Leu Ala Ile Leu Gln 420 425 430 Asp Tyr Pro SerLeu Arg Pro Pro Ile Asp His Leu Cys Glu Arg Leu 435 440 445 Pro Arg LeuGln Ala Arg Tyr Tyr Ser Ile Ala Ser Ser Ser Lys Val 450 455 460 His ProAsn Ser Val His Ile Cys Ala Val Val Val Glu Tyr Glu Thr 465 470 475 480Lys Ser Gly Arg Val Asn Lys Gly Val Ala Thr Ser Trp Leu Arg Ala 485 490495 Lys Glu Pro Ala Gly Glu Asn Gly Arg Arg Ala Leu Val Pro Met Phe 500505 510 Val Arg Lys Ser Gln Phe Arg Leu Pro Phe Lys Ala Thr Thr Pro Val515 520 525 Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Ile Gly PheIle 530 535 540 Gln Glu Arg Ala Trp Leu Gln Glu Gln Gly Lys Glu Val GlyGlu Thr 545 550 555 560 Leu Leu Tyr Tyr Gly Cys Arg Arg Ser Asp Glu AspTyr Leu Tyr Arg 565 570 575 Glu Glu Leu Ala Gln Phe His Ala Lys Gly AlaLeu Thr Arg Leu Ser 580 585 590 Val Ala Phe Ser Arg Glu Gln Pro Gln LysVal Tyr Val Gln His Leu 595 600 605 Leu Lys Arg Asp Lys Glu His Leu TrpLys Leu Ile His Asp Gly Gly 610 615 620 Ala His Ile Tyr Ile Cys Gly AspAla Arg Asn Met Ala Arg Asp Val 625 630 635 640 Gln Asn Thr Phe Cys AspIle Val Ala Glu Gln Gly Pro Met Glu His 645 650 655 Ala Gln Ala Val AspTyr Val Lys Lys Leu Met Thr Lys Gly Arg Tyr 660 665 670 Ser Leu Asp ValTrp Ser 675 57 19 DNA Bacteriophage SP6 primer 57 gatttaggtg acactatag19 58 44 DNA Artificial Sequence Synthetic adapter with NotI site andpoly dT tail 58 gactagttct agatcgcgag cggccgccct tttttttttt tttt 44 5916 DNA Artificial Sequence Top Strand of a SalI adapter 59 tcgacccacgcgtccg 16 60 12 DNA Artificial Sequence Bottom Strand of a SalI adapter,the first base is phosphorylated 60 gcctgcgcac cc 12 61 21 DNA humanoxidoreductase primer 1C 61 gtggaccaca agctcgtact g 21 62 22 DNA humanoxidoreductase primer 2C 62 catcgaccac ctgtgtgagc tg 22 63 22 DNA humanoxidoreductase primer 2D 63 gtacaggtag tcctcatccg ag 22 64 3710 DNAAspergillus niger NADP CYP450 oxidoreductaseZ26838 64 cctgtatcctgataactcct cagcaaatcg gagtaaacag aaggacaagt cattggagta 60 ctaagtagctccgtgtcaga gacccggaca ggatcagctt ctccgaaccc gagactccgg 120 gcgaaaaggccaccatcgct caggctacca cctgtgttcc ttccgtcgat cgtcctccct 180 cgtttccggctcacggcccc ccaaattatt gcggtctgct tagcagtggg ttcggcctct 240 ctgttcttcctggatcacac cacggcttac tttcttatcc ttttcctttt cctttcttcc 300 tttcttcctgttctcctttc ttcctttcca cccccttctt tcttttaacc ccatagcgtc 360 attctttcttccgttttatc tttggttttg ggacgccgcc accttatctc ggttcctgcc 420 tcggtctccggtgatcgcac ctggataggc taagcgtagg gaggtgtgac attcttcttt 480 cacctcctctccttttcccg cctcactccg ttcaatcccc cgctccaccc tttcagactc 540 gccatcgtatcaagtcgggg cctttgcttg cgccgctgaa cagcctcacc atggcgcaac 600 tcgataccctcgatctggtg gtcctggcgg tgcttttggt gggtagcgtg gcctacttca 660 ccaagggcacctactgggca gttgcaaaga cccgtatgcc tctaccggcc ccgcggatga 720 acggcgccgctaaggctggc aagactcgga acatcattga gaagatggaa gaaacgggca 780 agaattgtgttattttctac ggatcgcaaa ctggaaccgc tgaggactac gcctccagat 840 tggccaaggaaggatctcag cgcttcggcc tcaagaccat ggtggctgac ctcgaggaat 900 acgactatgagaacctggac caattcccgg aggacaaggt tgcgtttttc gtgctcgcca 960 cctacggagagggtgagcct acggataatg ctgttgagtt ctaccagttc ttcaccggtg 1020 acgacgttgcttttgagagc gcgtccgcgg acgagaagcc tctgtccaag ctgaagtatg 1080 ttgctttcggtctgggtaac aacacttatg agcactacaa cgccatggtt cgtcaagtcg 1140 atgctgctttccagaagctc gggccgcagc gtattggttc tgctggcgag ggtgatgacg 1200 gtgccggtacaatggaagaa gacttcttgg cctggaagga gcccatgtgg gcagcactgt 1260 cggagtcgatggatctcgaa gagcgtgaag cggtctacga acctgttttc tgcgtcaccg 1320 aaaacgagtccctgagccct gaggacgaga cggtctatct tggagagccc acccagagcc 1380 accttcagggtactcccaaa ggcccgtact ctgcgcacaa cccctttatc gcccctattg 1440 ccgaatctcgtgagcttttc accgtcaagg atcgcaactg tctgcacatg gaaattagca 1500 tcgctggaagtaacttgtcc taccagactg gtgaccacat cgctgtttgg cccacaaacg 1560 ctggtgccgaagtggatcgg ttccttcagg tcttcggtct cgagggcaag cgtgattcgg 1620 tcatcaacatcaagggtatc gatgttacgg ccaaggtccc aatcccgacc ccgaccacgt 1680 acgatgccgctgttcggtac tatatggaag tctgcgcccc tgtgtcccgt cagtttgtag 1740 ccactctggccgcgttcgct ccgatgagga aagcaaggca gagattgtgc gtctgggtag 1800 cacaaggactatttccacga gaaggtcacc aaccaatgct tcaacatgcc caggctcttc 1860 agagcatcacgtccaagcct ttctctgctg ttccgttctc tctgcttatt gaaggcatta 1920 cgaagctgcagcctcgctac tactcgatct cttcgtcctc ccttgtccag aaggacaaga 1980 tcagcatcacggccgttgtg gaatctgttc gtctgcccgg tgcctctcac atggtgaagg 2040 gtgtgactacgaattatctc ctcgcgctca agcagaagca gaacgggcga tccctctccc 2100 gaccctcacggcttgactta ctccatcacg gtccccggaa caagtacgac ggtatccacg 2160 ttcccgtgcatgttcgccac tcgaacttca agctgccctc tgatccctct cggcccatta 2220 tcatggttggtcctggtact ggtgttgctc ctttccgtgg tttcattcag gaacgtgctg 2280 ctttggcggccaagggcgag aaggttggac ccactgttct cttcttcggt tgccgcaaga 2340 gtgacgaggatttcttgtac aaggatgaat ggaaggtaag atatcttttt ttcttttccg 2400 cagctaccttcatacatctc ggatgctaac atatcgcgat tcgcagacct atcaggacca 2460 gcttggagacaacttgaaga tcatcactgc gttctcgcgt gagggtcctc agaaggtcta 2520 cgttcagcacagactccgcg agcactccga acttgtcagc gaccttctga agcagaaagc 2580 taccttctacgtctgtggtg acgctgcaaa catggctcgc gaggttaacc ttgtgcttgg 2640 ccagatcattgctgcgcagc gtggtctgcc cgccgagaag ggcgaagaaa tggtcaagca 2700 catgcgtagacgtggacgct accaggaaga tgtgtggtca taatctttca atgcatcgac 2760 ttttctttcttgtctatcac gacggccttc tcgatccatt attttattta acgcctagat 2820 gatctttgcatatatactcc gctgattttg cctattcatc tgttttgctt ggcgtggttt 2880 atgtatgcctagtttatttg ttttgtgcac cgaccggcca gccacacatt gaagtggctt 2940 gagcatgagtgcggtagcca gtgtcgaaag aacaggatag acgatcatga ttattgcggg 3000 aacatgttatgccattctgg gcatattgat atctggttgc atgagcccag aggatacgaa 3060 aagatgaatccatatttaat ttgcacaata cttttcgcct tcttcatcta gtaattaaat 3120 taattgagcactgaccgaac gagctgacac ctgctgctcg gaatagccga caacgcattg 3180 acgtgcaagagatgcataat cattacaatc aacaagtaga ctggtaacta aatcactgaa 3240 tactacagttactgcctact ttcagccaaa aagtaatact gaagatttcg gggaatcaaa 3300 tagaagaaacatgcataagc ccaacctcgg caataccggg agttaagcac agtaaccaaa 3360 accaaaccaaactagaaccg gcgcgcgacc agtgacccat cgtcattccc ggtatcagca 3420 gttcagtcagactggctggc tagcccgaac ccaactgccg caatcatcca tccatcctca 3480 acccgcccctcccatgccaa cctctctact ccgcagagcg agggacaaaa aaatgagatg 3540 cagcaattaaccacgataat ctagcaaaaa gaaagttaga agccggaaga acatacatat 3600 cgcttttaccgctgttcgac tgcgacgacg ggtcttgaga gcagttccgc cacgtgggcg 3660 aaaagctggactgcacacta cttacgctac cctacgctac ctcggtaccc 3710 65 693 PRT Aspergillusniger NADPCYP450oxidoreductaseCAA81550 65 Met Ala Gln Leu Asp Thr LeuAsp Leu Val Val Leu Ala Val Leu Leu 1 5 10 15 Val Gly Ser Val Ala TyrPhe Thr Lys Gly Thr Tyr Trp Ala Val Ala 20 25 30 Lys Thr Arg Met Pro LeuPro Ala Pro Arg Met Asn Gly Ala Ala Lys 35 40 45 Ala Gly Lys Thr Arg AsnIle Ile Glu Lys Met Glu Glu Thr Gly Lys 50 55 60 Asn Cys Val Ile Phe TyrGly Ser Gln Thr Gly Thr Ala Glu Asp Tyr 65 70 75 80 Ala Ser Arg Leu AlaLys Glu Gly Ser Gln Arg Phe Gly Leu Lys Thr 85 90 95 Met Val Ala Asp LeuGlu Glu Tyr Asp Tyr Glu Asn Leu Asp Gln Phe 100 105 110 Pro Glu Asp LysVal Ala Phe Phe Val Leu Ala Thr Tyr Gly Glu Gly 115 120 125 Glu Pro ThrAsp Asn Ala Val Glu Phe Tyr Gln Phe Phe Thr Gly Asp 130 135 140 Asp ValAla Phe Glu Ser Ala Ser Ala Asp Glu Lys Pro Leu Ser Lys 145 150 155 160Leu Lys Tyr Val Ala Phe Gly Leu Gly Asn Asn Thr Tyr Glu His Tyr 165 170175 Asn Ala Met Val Arg Gln Val Asp Ala Ala Phe Gln Lys Leu Gly Pro 180185 190 Gln Arg Ile Gly Ser Ala Gly Glu Gly Asp Asp Gly Ala Gly Thr Met195 200 205 Glu Glu Asp Phe Leu Ala Trp Lys Glu Pro Met Trp Ala Ala LeuSer 210 215 220 Glu Ser Met Asp Leu Glu Glu Arg Glu Ala Val Tyr Glu ProVal Phe 225 230 235 240 Cys Val Thr Glu Asn Glu Ser Leu Ser Pro Glu AspGlu Thr Val Tyr 245 250 255 Leu Gly Glu Pro Thr Gln Ser His Leu Gln GlyThr Pro Lys Gly Pro 260 265 270 Tyr Ser Ala His Asn Pro Phe Ile Ala ProIle Ala Glu Ser Arg Glu 275 280 285 Leu Phe Thr Val Lys Asp Arg Asn CysLeu His Met Glu Ile Ser Ile 290 295 300 Ala Gly Ser Asn Leu Ser Tyr GlnThr Gly Asp His Ile Ala Val Trp 305 310 315 320 Pro Thr Asn Ala Gly AlaGlu Val Asp Arg Phe Leu Gln Val Phe Gly 325 330 335 Leu Glu Gly Lys ArgAsp Ser Val Ile Asn Ile Lys Gly Ile Asp Val 340 345 350 Thr Ala Lys ValPro Ile Pro Thr Pro Thr Thr Tyr Asp Ala Ala Val 355 360 365 Arg Tyr TyrMet Glu Val Cys Ala Pro Val Ser Arg Gln Phe Val Ala 370 375 380 Thr LeuAla Ala Phe Ala Pro Met Arg Lys Ala Arg Gln Arg Leu Cys 385 390 395 400Val Trp Val Ala Gln Gly Leu Phe Pro Arg Glu Gly His Gln Pro Met 405 410415 Leu Gln His Ala Gln Ala Leu Gln Ser Ile Thr Ser Lys Pro Phe Ser 420425 430 Ala Val Pro Phe Ser Leu Leu Ile Glu Gly Ile Thr Lys Leu Gln Pro435 440 445 Arg Tyr Tyr Ser Ile Ser Ser Ser Ser Leu Val Gln Lys Asp LysIle 450 455 460 Ser Ile Thr Ala Val Val Glu Ser Val Arg Leu Pro Gly AlaSer His 465 470 475 480 Met Val Lys Gly Val Thr Thr Asn Tyr Leu Leu AlaLeu Lys Gln Lys 485 490 495 Gln Asn Gly Arg Ser Leu Ser Arg Pro Ser ArgLeu Asp Leu Leu His 500 505 510 His Gly Pro Arg Asn Lys Tyr Asp Gly IleHis Val Pro Val His Val 515 520 525 Arg His Ser Asn Phe Lys Leu Pro SerAsp Pro Ser Arg Pro Ile Ile 530 535 540 Met Val Gly Pro Gly Thr Gly ValAla Pro Phe Arg Gly Phe Ile Gln 545 550 555 560 Glu Arg Ala Ala Leu AlaAla Lys Gly Glu Lys Val Gly Pro Thr Val 565 570 575 Leu Phe Phe Gly CysArg Lys Ser Asp Glu Asp Phe Leu Tyr Lys Asp 580 585 590 Glu Trp Lys ThrTyr Gln Asp Gln Leu Gly Asp Asn Leu Lys Ile Ile 595 600 605 Thr Ala PheSer Arg Glu Gly Pro Gln Lys Val Tyr Val Gln His Arg 610 615 620 Leu ArgGlu His Ser Glu Leu Val Ser Asp Leu Leu Lys Gln Lys Ala 625 630 635 640Thr Phe Tyr Val Cys Gly Asp Ala Ala Asn Met Ala Arg Glu Val Asn 645 650655 Leu Val Leu Gly Gln Ile Ile Ala Ala Gln Arg Gly Leu Pro Ala Glu 660665 670 Lys Gly Glu Glu Met Val Lys His Met Arg Arg Arg Gly Arg Tyr Gln675 680 685 Glu Asp Val Trp Ser 690

1. An isolated and purified nucleic acid, encoding an Aspergillusochraceus 11 alpha hydroxylase.
 2. An isolated and purified nucleic acidof claim 1, wherein said nucleic acid comprises the DNA sequence of SEQID NO:
 1. 3. An isolated and purified Aspergillus ochraceus 11 alphahydroxylase.
 4. An isolated and purified Aspergillus ochraceus 11 alphahydroxylase of claim 3 comprising an amino acid sequence of SEQ ID NO:2.
 5. A fusion protein comprising the amino acid sequence of theAspergillus ochraceus 11 alpha hydroxylase of claim
 4. 6. An isolatedand purified nucleic acid, encoding an Aspergillus ochraceusoxidoreductase.
 7. An isolated and puified nucleic acid of claim 6,wherein said nucleic acid comprises the DNA sequence of SEQ ID NO:
 5. 8.An isolated and purified Aspergillus ochraceus oxidoreductase.
 9. Anisolated and purified Aspergillus ochraceus oxidoreductase of claim 8comprising an amino acid sequence of SEQ ID NO:
 6. 10. An isolated andpurified nucleic acid encoding an enzyme that can catalyze the 11 alphahydroxylation of a compound selected from the group consisting of: 3keto delta 4,5 steroids (3 keto delta 4 steroids); 3 keto delta 4,5delta 6,7 steroids (3 keto delta 4 delta 6 steroids); 3 keto delta 6,7steroids (3 keto delta 6 steroids); and 3 keto delta 1,2 delta 4,5steroids (3 keto delta 1 delta 4 steroids).
 11. An isolated and purifiednucleic acid of claim 10, wherein said enzyme does not catalyze the 15beta hydroxylation of a compound selected from the group consisting of:3 keto delta 4,5 steroids; 3 keto delta 4,5 delta 6,7 steroids; and 3keto delta 6,7 steroids.
 12. The isolated and purified nucleic acid ofclaim 10 or claim 11, wherein said hydroxylation is selected from thegroup consisting of: (a) canrenone to 11 alpha hydroxy canrenone; (b)androstenedione to 11 alpha hydroxy androstenedione; (c) aldona to 11alpha hydroxy aldona; (d) ADD (1,4 androstenedienedione) to 11 alphahydroxy ADD; (e) mexrenone to 11 alpha hydroxy mexrenone; (f) 6 betamexrenone to 11 alpha hydroxy 6 beta mexrenone; (g) 9 alpha mexrenone to11 alpha hydroxy 9 alpha mexrenone; (h) 12 beta mexrenone to 11 alphahydroxy 12 beta mexrenone; (i) delta 12 mexrenone to 11 alpha hydroxydelta 12 mexrenone; (j) testosterone to 11 alpha hydroxy testosterone;(k) progesterone to 11 alpha hydroxy progesterone; (l) mexrenone6,7-bis-lactone to 11 alpha hydroxy mexrenone 6,7-bis-lactone; and (m)mexrenone 7,9-bislactone to 11 alpha hydroxy mexrenone 7,9-bislactone.13. The isolated and purified nucleic acid of claim 12, wherein saidhydroxylation is selected from the group consisting of: (a) canrenone to11 alpha hydroxy canrenone; (b) androstenedione to 11 alpha hydroxyandrostenedione; (c) aldona to 11 alpha hydroxy aldona; and (d) ADD (1,4androstenedienedione) to 11 alpha hydroxy ADD.
 14. The isolated andpurified nucleic acid of claim 13, wherein said hydroxylation is fromcanrenone to 11 alpha hydroxy canrenone.
 15. A method of expressing aprotein that can catalyze the 11 alpha hydroxylation of a compoundselected form the group consisting of: 3 keto delta 4,5 steroids; 3 ketodelta 4,5 delta 6,7 steroids; 3 keto delta 6,7 steroids; and 3 ketodelta 1,2 delta 4,5 steroids comprising; (a) transforming ortransfecting host cells with an expression cassette comprising apromoter operably linked to a nucleic acid that encodes said protein,and (b) expressing said protein in said host cells.
 16. A method ofproducing the protein of claim 15, further comprising the step ofrecovering said protein.
 17. The method of claim 15 or claim 16 whereinsaid protein is an Aspergillus ochraceus 11 alpha hydroxylase.
 18. Themethod of claim 17, further comprising expressing an electron donorprotein, wherein said electron donor protein can donate electrons tosaid protein that can catalyze the 11 alpha hydroxylation of a compoundselected from the group consisting of: 3 keto delta 4,5 steroids; 3 ketodelta 4,5 delta 6,7 steroids; 3 keto delta 6,7 steroids; and 3 ketodelta 1,2 delta 4,5 steroids.
 19. The method of claim 18 wherein saidelectron donor protein is selected from the group consisting of humanoxidoreductase and Aspergillus ochraceus oxidoreductase.
 20. The methodof claim 18 wherein said electron donor protein is Aspergillus ochraceusoxidoreductase.
 21. The method of claim 18, wherein the nucleic acidencoding said 11 alpha hydroxylase and said electron donor protein areon separate expression cassettes.
 22. The method of claim 18, whereinthe nucleic acid encoding said 11 alpha hydroxylase and said electrondonor protein are on the same expression cassettes.
 23. The method ofclaim 21 wherein said 11 alpha hydroxylase is Aspergillus ochraceus 11alpha hydroxylase and said electron donor protein is humanoxidoreductase.
 24. The method of claim 22 wherein said 11 alphahydroxylase is Aspergillus ochraceus 11 alpha hydroxylase and saidelectron donor protein is human oxidoreductase. 25 The method of claim21 wherein said 11 alpha hydroxylase is Aspergillus ochraceus 11 alphahydroxylase and said electron donor protein is Aspergillus ochraceusoxidoreductase. 26 The method of claim 22 wherein said 11 alphahydroxylase is Aspergillus ochraceus 11 alpha hydroxylase and saidelectron donor protein is Aspergillus ochraceus oxidoreductase
 27. Themethod of claim 17, wherein said Aspergillus ochraceus 11 alphahydroxylase is SEQ ID NO:
 2. 28. The method of claim 19, wherein saidhuman oxidoreductase is SEQ ID NO:
 4. 29. The method of claim 19,wherein said Aspergillus ochraceus oxidoreductase is SEQ ID NO:
 6. 30.An isolated and purified polypeptide that can catalyze the 11 alphahydroxylation of a compound selected from the group consisting of: 3keto delta 4,5 steroids (3 keto delta 4 steroids); 3 keto delta 4,5delta 6,7 steroids (3 keto delta 4 delta 6 steroids); 3 keto delta 6,7steroids (3 keto delta 6 steroids); and 3 keto delta 1,2 delta 4,5steroids (3 keto delta 1 delta 4 steroids).
 31. An isolated and purifiedpolypeptide claim 30, wherein said enzyme does not catalyze the 15 betahydroxylation of a compound selected from the group consisting of: 3keto delta 4,5 steroids; 3 keto delta 4,5 delta 6,7 steroids; and 3 ketodelta 6,7 steroids.
 32. The isolated and purified polypeptide of claim30 or claim 31, wherein said hydroxylation is selected from the groupconsisting of: (a) canrenone to 11 alpha hydroxy canrenone; (b)androstenedione to 11 alpha hydroxy androstenedione; (c) aldona to 11alpha hydroxy aldona; (d) ADD (1,4 androstenedienedione) to 11 alphahydroxy ADD; (e) mexrenone to 11 alpha hydroxy mexrenone; (f) 6 betamexrenone to 11 alpha hydroxy 6 beta mexrenone; (g) 9 alpha mexrenone to11 alpha hydroxy 9 alpha mexrenone; (h) 12 beta mexrenone to 11 alphahydroxy 12 beta mexrenone; (i) delta 12 mexrenone to 11 alpha hydroxydelta 12 mexrenone; (j) testosterone to 11 alpha hydroxy testosterone;(k) progesterone to 11 alpha hydroxy progesterone; (l) mexrenone6,7-bis-lactone to 11 alpha hydroxy mexrenone 6,7-bis-lactone; and (m)mexrenone 7,9-bislactone to 11 alpha hydroxy mexrenone 7,9-bislactone.33. The isolated and purified polypeptide of claim 32, wherein saidhydroxylation is selected from the group consisting of: (a) canrenone to11 alpha hydroxy canrenone; (b) androstenedione to 11 alpha hydroxyandrostenedione; (c) aldona to 11 alpha hydroxy aldona; and (d) ADD (1,4androstenedienedione) to 11 alpha hydroxy ADD.
 34. The isolated andpurified enzyme of claim 33, wherein said hydroxylation is fromcanrenone to 11 alpha hydroxy canrenone.
 35. An expression cassettecomprising a promoter operably linked to an isolated and purifiednucleic acid of claim 10 encoding a polypeptide that can catalyze the 11alpha hydroxylation of a compound selected from the group consisting of:3 keto delta 4,5 steroids (3 keto delta 4 steroids); 3 keto delta 4,5delta 6,7 steroids (3 keto delta 4 delta 6 steroids); 3 keto delta 6,7steroids (3 keto delta 6 steroids); and 3 keto delta 1,2 delta 4,5steroids (3 keto delta 1 delta 4 steroids).
 36. An expression cassettecomprising a promoter operably linked to an isolated and purifiednucleic acid of claim 6 encoding Aspergillus ochraceus oxidoreductase.37. An expression cassette of claim 36 wherein said nucleic acid is SEQID NO:
 06. 38. An expression cassette comprising a heterologous DNAencoding an enzyme from the metabolic pathway for the synthesis ofsitosterol to eplerenone wherein said enzyme catalyzes at least oneconversion selected from the group consisting of: (a) canrenone to 11alpha hydroxy canrenone; (b) androstenedione to 11 alpha hydroxyandrostenedione; (c) aldona to 11 alpha hydroxy aldona; (d) ADD (1,4androstenedienedione) to 11 alpha hydroxy ADD; (e) mexrenone to 11 alphahydroxy mexrenone; (f) 6 beta mexrenone to 11 alpha hydroxy 6 betamexrenone; (g) 9 alpha mexrenone to 11 alpha hydroxy 9 alpha mexrenone;(h) 12 beta mexrenone to 11 alpha hydroxy 12 beta mexrenone; (i) delta12 mexrenone to 11 alpha hydroxy delta 12 mexrenone; (j) testosterone to11 alpha hydroxy testosterone; (k) progesterone to 11 alpha hydroxyprogesterone; (l) mexrenone 6,7-bis-lactone to 11 alpha hydroxymexrenone 6,7-bis-lactone; (m) mexrenone 7,9-bislactone to 11 alphahydroxy mexrenone 7,9-bislactone; and wherein the heterologous DNA isoperably linked to control sequences required to express the encodedenzymes in a recombinant host.
 39. The expression cassette according toclaim 38, characterized in that the heterologous DNA coding sequencesare selected from the group consisting of the following genus andspecies: Aspergillus ochraceus, Aspergillus ochraceus, Aspergillusniger, Aspergillus nidulans, Rhizopus oryzae, Rhizopus stolonifer,Streptomyces fradiae, Bacillus megaterium, Pseudomonas cruciviae,Trichothecium roseum, Fusarium oxysporum Rhizopus arrhizus, Absidiacoerula, Absidia glauca, Actinomucor elegans, Aspergillus flavipes,Aspergillus fumigatus, Beauveria bassiana, Botryosphaeria obtusa,Calonectria decora, Chaetomium cochliodes, Corynespora cassiicola,Cunninghamella blakesleeana, Cunninghamella echinulata, Cunninghamellaelegans, Curvularia clavata, Curvularia lunata, Cylindrocarponradicicola, Epicoccum humicola, Gongronella butleri, Hypomyceschrysospermus, Monosporium olivaceum, Mortierella isabellina, Mucormucedo, Mucor griseocyanus, Myrothecium verrucaria, Nocardia corallina,Paecilomyces carneus, Penicillum patulum, Pithomyces atroolivaceus,Pithomyces cynodontis, Pycnosporium sp., Saccharopolyspora erythrae,Sepedonium chrysospermum, Stachylidium bicolor, Streptomyceshyqroscopicus, Streptomyces purpurascens, Syncephalastrum racemosum,Thamnostylum piriforme, Thielavia terricola, and Verticilliumtheobrornae, Cephalosporium aphidicola, Cochliobolus lunatas,Tieghemella orchidis, Tieghemella hyalospora, Monosporium olivaceum,Aspergillus ustus, Fusarium graminearum, Verticillium glaucum, andRhizopus nigricans.
 40. The expression cassette according to claim 39,wherein the genus and species are selected from the group consisting ofAspergillus ochraceus, Aspergillus ochraceus, Aspergillus niger,Aspergillus nidulans, Rhizopus oryzae, Rhizopus stolonifer, Streptomycesfradiae, Bacillus megaterium, Pseudomonas cruciviae, Trichotheciumroseum, Fusarium oxysporum Rhizopus arrhizus, and Monosporium olivaceum.41. The expression cassette according to claim 40, wherein the genusspecies is Aspergillus ochraceus.
 42. A recombinant host cell andprogeny thereof comprising at least one expression cassette according toclaim
 38. 43. A process for making one or more enzymes from themetabolic pathway for the synthesis of sitosterol to eplerenonecomprising incubating the recombinant host cell of claim 42 in anutrient medium under conditions where the one or more enzymes encodedby the heterologous DNA are expressed and accumulate.
 44. A process forthe selective oxidation of a compound to an hydroxylated product, whichprocess comprises the steps of: (a) incubating the compound to behydroxylated in the presence the recombinant host cells of claim 42under conditions where the compound is hydroxylated and the hydroxylatedproduct accumulates, and (b) recovering the hydroxylated product.
 45. Aprocess for the selective hydroxylation of a compound to an hydroxylatedproduct in vitro, which process comprises the steps of: (a) incubatingthe compound to be hydroxylated in the presence of the enzymes producedin the process of claim 44 under conditions where the compound ishydroxylated and the hydroxylated product accumulates, and (b)recovering the hydroxylated product.
 46. A host cell harboring anexpression cassette of any of claim
 35. 47. A host cell of claim 46,wherein said expression cassette is integrated into the chromosome ofsaid host cell.
 48. A host cell of claim 46, wherein said expressioncassette is integrated into an expression vector.
 49. A method ofdetermining the specific activity of a cloned 11 alpha hydroxylasecomprising the steps of; (a) transforming host cells with an expressionvector comprising a nucleic acid that encodes said 11 alpha hydroxylase,(b) expressing said 11 alpha hydroxylase in said host cells; (c)preparing subcellular membrane fractions from said cells, (d) incubatingsaid subcellular membrane fractions microsomes with a steroid substrate,and (e) monitoring conversion of the steroid substrate to its 11 alphahydroxy steroid counterpart.
 50. An isolated and purified antibodyhaving a binding specificity for 11 alpha hydroxylase of claim 3 asshown in SEQ ID NO:
 2. 51. An isolated and purified antibody having abinding specificity for the 11 alpha hydroxylase of claim
 4. 52. Theantibody of claim 51 which binds to a protein region selected from thegroup consisting of (a) the N-terminal amino acids 1-10 of SEQ ID NO: 2;(b) the last 10 C-terminal amino acids of SEQ ID NO: 2; (c) SEQ ID NO:23; (d) SEQ ID NO: 24; and (e) SEQ ID NO:
 25. 53. An isolated andpurified antibody having a binding specificity for oxidoreductase ofclaim 8
 54. An isolated and purified antibody having a bindingspecificity for the oxidoreductase of claim
 9. 55. The antibody of claim54 which binds to a protein region selected from the group consistingof: (a) the N-terminal amino acids 1-10 of SEQ ID NO: 6; (b) the last 10C-terminal amino acids of SEQ ID NO: 6; and (c) SEQ ID NO:
 26. 56. Acomposition comprising the antibody of claim 50 or 53 and an effectivecarrier, vehicle, or auxiliary agent.
 57. An isolated nucleic acid thatspecifically hybridizes under low stringent conditions to the nucleicacid of claim 2
 58. An isolated nucleic acid that specificallyhybridizes under high stringent conditions to the nucleic acid of claim2.
 59. An isolated nucleic acid that specifically hybridizes underlowstringent conditions to the nucleic acid of claim 7
 60. An isolatednucleic acid that specifically hybridizes under high stringentconditions to the nucleic acid of claim
 7. 61. Use of a host cellharboring a cloned 11 alpha hydroxylase of claim 1 for the manufactureof a medicament for therapeutic application to treat heart disease,inflammation, arthritis, or cancer.
 62. A composition comprising fromabout 0.5-500 g/L molasses, 0.5-50 g/L cornsteep liquid, 0.5-50 g/LKH₂PO₄, 2.5-250 g/L NaCl, 2.5-250 g/L glucose, and 0.04-4 g/Lprogesterone, pH 3.5-7.
 63. The composition of claim 62 comprising fromabout 10-250 g/L molasses, 1-25 g/L cornsteep liquid, 1-25 g/L KH₂PO₄,5-125 g/L NaCl, 5-125 g/L glucose, and 0.08-2 g/L progesterone, pH4.5-6.5.
 64. The composition of claim 62 comprising from about 25-100g/L molasses, 2.5-10 g/L cornsteep liquid, 2.5-10 g/L KH₂PO₄, 12.5-50g/L NaCl, 12.5-50 g/L glucose, and 0.2-0.8 g/L progesterone, pH 5.5-6.0.65. The composition of claim 62 comprising from 50 g/L molasses, 5 g/Lcornsteep liquid, 5 g/L KH₂PO₄, 25 g/L NaCl, 25 g/L glucose, 20 g/Lagar, and 0.4 g/L progesterone, pH 5.8.
 66. A composition of claim 62further comprising from about 4-100 g/L agar.
 67. A composition of claim66 further comprising from about 10-40 g/L agar.
 68. A composition ofclaim 66 further comprising about 20 g/L agar.
 69. Use of thecomposition of claim 62 or 66 to produce spores from the microorganismselected from the group consisting of Aspergillus ochraceus, Aspergillusniger, Aspergillus nidulans, Rhizopus oryzae, Rhizopus stolonifer, andTrichothecium roseum, Fusarium oxysporum Rhizopus arrhizus, Monosporiumolivaceum. Penicillum chrysogenum, and Absidia coerula.
 70. Use of thecomposition of any of claims 62 or 66 to produce spores from Aspergillusochraceus.
 71. A fusion protein comprising the amino acid sequence ofthe Aspergillus ochraceus oxidoreductase of claim
 8. 72. An isolated andpurified Aspergillus ochraceus 11 alpha hydroxylase encoded by thenucleic acid of claim
 57. 73. An isolated and purified Aspergillusochraceus 11 alpha hydroxylase encoded by the nucleic acid of claim 58.74. An isolated and purified Aspergillus ochraceus oxidoreductaseencoded by the nucleic acid of claim
 59. 75. An isolated and purifiedAspergillus ochraceus oxidoreductase encoded by the nucleic acid ofclaim
 60. 76. A method of converting a steroid to its 11 alphahydroxylated form comprising the steps of: a.) contacting the steroidwith the Aspergillus ochraceus 11 alpha hydroxylase of claim 3
 77. A 11alpha hydroxylated steroid made by the method of claim 76.